Oral compositions of mk2 pathway inhibitor for treatment of immune conditions

ABSTRACT

The present disclosure relates to oral compositions of Compound I or a derivative thereof. Methods of use for treating an inflammatory condition are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 63/000,746, filed on Mar. 27, 2020; Ser. No.63/015,241, filed on Apr. 24, 2020; Ser. No. 63/018,954, filed on May 1,2020; Ser. No. 63/022,301, filed on May 8, 2020, Ser. No. 63/022,298,filed on May 8, 2020, Ser. No. 63/024,160 filed on May 13, 2020, Ser.No. 63/053,903, filed on Jul. 20, 2020, Ser. No. 63/076,689, filed onSep. 10, 2020, Ser. No. 63/126,173, filed on Dec. 16, 2020, Ser. No.63/128,523, filed on Dec. 21, 2020; Ser. No. 63/136,080, filed on Jan.11, 2021, Ser. No. 63/136,967, filed on Jan. 13, 2021, Ser. No.63/138,672, filed on Jan. 18, 2021, Ser. No. 63/140,116, filed on Jan.21, 2021, and Ser. No. 63/149,230, filed on Feb. 13, 2021, each of whichis hereby incorporated by reference in its entirety.

SUMMARY

The present disclosure is directed to a method of treating aninflammatory condition comprising administering to a human subjecthaving an inflammatory condition, an oral dose of 5 mg/day to 300 mg/dayof Compound I having the following structure:

or a derivative thereof to treat said inflammatory condition.

The present disclosure is further directed to oral pharmaceuticalcompositions comprising Compound I or a derivative thereof, where theoral compositions comprise 5 mg to 300 mg of Compound I and apharmaceutically acceptable carrier.

As used throughout this disclosure, recitation of “Compound I”encompasses atropisomer compounds (P)-I and (M)-I as disclosed below inany molar ratio from 4:1 ((P)-I:(M)-I) to 999:1, and also includesembodiments where Compound (P)-I is substantially free from Compound(M)-I. Compounds (P)-I and (M)-I can be in any form (e.g., free base,crystalline form, etc.) as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the mean (SD) plasma concentration-timeprofiles of ATI-450 after a single dose under fasted conditions,semi-log scale.

FIG. 2 is a graph showing mean (SD) plasma concentration-time profilesof ATI-450 after 7 days of BID (twice daily) dosing under fastedconditions, semi-log scale.

FIG. 3 shows ATI-450 modulation of ex vivo LPS stimulatedcytokine/chemokine production in blood samples taken from subjects dosedwith placebo or 50 mg/BID ATI-450.

FIGS. 4A-4E are graphs showing ex vivo LPS stimulated production ofpHSP27 (FIG. 4A, top), TNF-α (FIG. 4B, top), IL-1β (FIG. 4C, top), IL-8(FIG. 4D, top), IL-6 (FIG. 4E, top) in Day 7 blood samples taken fromsubjects dosed with 10 mg, 30 mg, 50 mg, 80 mg, and 120 mg of ATI-450 orplacebo. FIGS. 4A-4E also show mean (±SEM) levels of pHSP27 (FIG. 4A,bottom), TNF-α (FIG. 4B, bottom), IL-1β (FIG. 4C, bottom), IL-8 (FIG.4D, bottom), IL-6 (FIG. 4E, bottom) in subjects administered 10 mg, 30mg, 50 mg, 80 mg, and 120 mg twice daily, comparing day 1 pre-dosingvalues (set to 100%) with day 7 values 4 hours after dosing (approximateC_(max)) and 12 hours after dosing (C_(trough)).

FIG. 5 is a graph showing differential modulation of ex vivo stimulatedIL-10 versus TNF-α and IL-1β in blood samples taken from subjects dosedwith placebo or 80 mg or 120 mg of ATI-450.

FIG. 6 depicts three graphs comparing ATI-450 modulation of LPS andIL-1β stimulated TNF-α (far left), IL-6 (middle), and IL-8 (far right)production.

FIGS. 7A-7C show pharmacokinetic (PK) data of ATI-450 followingadministration of at 80 mg or 120 mg BID. The graphs of FIG. 7A showmean plasma concentration-time profiles of ATI-450 after 1 day (top) and7 days (middle and bottom) of BID dosing, semi-log scale. FIG. 7B aregraphs showing mean (+/−std. dev.) plasma concentration-time profiles ofATI-450 dosed at 120 mg BID. FIG. 7C are graphs showing the plasmaconcentration-time profiles of ATI-450 in individual subjects at days 1(left) and 7 (right) following 120 mg BID dosing.

FIG. 8 is a graph showing the median change in DAS28-CRP from baselinefor all patients in placebo and ATI-450 (50 mg/BID) treatment groups atdays 1, 7, 14, 28, 42, 56, and 84 of treatment and after treatment endedat day 112. The numbers on each line represent the number of patients ateach timepoint.

FIG. 9 is a graph showing the percent of patients having DAS-28 <2.6 and<3.2 over the course of treatment (through day 84).

FIG. 10 is a graph showing the median percent change in high sensitivityC-reactive protein (hsCRP) (mg/mL) from baseline for all patient inplacebo and ATI-450 (50 mg/BID) treatment groups at days 1, 7, 14, 28,42, 56, and 84 of treatment and after treatment ended at day 112. Thenumbers on each line represent the number of patients at each timepoint.

FIG. 11 is a graph showing the median percent change in swollen jointcount from baseline for all patients in placebo and ATI-450 (50 mg/BID)treatment groups at days 1, 7, 14, 28, 42, 56, and 84 of treatment andafter treatment ended at day 112. The numbers on each line represent thenumber of patients at each timepoint.

FIG. 12 is a graph showing the median percent change in tender jointcount from baseline for all patients in placebo and ATI-450 (50 mg/BID)treatment groups at days 1, 7, 14, 28, 42, 56, and 84 of treatment andafter treatment ended at day 112. The numbers on each line represent thenumber of patients at each timepoint.

FIGS. 13A-13D are graphs showing median percent change in patient visualanalog scale (VAS) for disease activity (FIG. 13A), patient VAS forarthritic pain (FIG. 13B), Health Assessment Questionnaire (HAQ)Disability Index (DI) (FIG. 13C), and physician VAS scores for diseaseactivity (FIG. 13C).

FIG. 14 shows the ACR20/50/70 response over the course of the study(through day 112) for placebo and ATI-450 (50 mg/BID) treatment groups.

FIG. 15 is a graph showing the percent of patients responsive to ATI-450treatment as assessed by the Hand-Wrist MRI RAMRIS. The RAMRIS providessub-scores for CARLOS (cartilage loss), erosion, osteitis, synovitis,and the graph of FIG. 15 is showing percent responsiveness in theseendpoints by treatment and hand. A patient was deemed responsive if atleast a 1-point improvement in any hand was observed.

FIG. 16 is a graph showing the change in baseline at day 84 for CARLOS,erosion, osteitis, and synovitis endpoints in placebo and ATI-450 (50 mgBID) patients. The data represents the average of both hands as assessedby the Hand-Wrist MRI RAMRIS. Diamonds represent mean values.

FIG. 17 is a graph showing the change in baseline at day 84 for CARLOS,erosion, osteitis, and synovitis endpoints for each hand in placebo andATI-450 (50 mg BID) patients. The data the average of each hand asassessed by the Hand-Wrist MRI RAMRIS. Diamonds represent mean values.

FIGS. 18A-18B are a graphs showing ex vivo LPS-stimulated cytokines,including TNF-α and IL-1β (FIG. 18A) and IL-6 and IL-8 (FIG. 18B), atday 1 versus day 84 in blood samples from patient in placebo and ATI-450(50 mg/BID) treatment groups.

FIGS. 19A-19B show the impact of ATI-450 treatment (50 mg/BID) onendogenous plasma levels of the pro-inflammatory cytokine, TNF-α (FIG.19A), and the anti-inflammatory cytokine, IL-1RA (FIG. 19B), at days 1,28, 56, and 84 of treatment.

FIGS. 20A-20D shows the impact of ATI-450 treatment (50 mg/BID) onendogenous plasma levels of TNF-α (FIG. 20A), IL-8 (FIG. 20B), IL-6(FIG. 20C), and MIP1β (FIG. 20D) at days 1, 28, 56, and 84 of treatment.

FIG. 21 is a graph showing ATI-450 mediated increase in T regulatory(Treg) cells in a murine collagen-induced arthritis model. The timelineabove the graph depicts timing and dose of ATI-450 and ENBREL® (tumornecrosis factor inhibitor) in mice following collagen administration.Treg cells were identified as Foxp3⁺/CD4⁺ cells.

FIGS. 22A-22C are graphs showing ATI-450 inhibition of TLR4 mediatedcytokine release (FIG. 22A), TLR3 mediated cytokine release (FIG. 22B),and TLR7,8 mediated cytokine release (FIG. 22C).

FIG. 23 is a graph showing that ATI-450 dose-dependently inhibits TLR2mediated induction of TNF-α production.

FIG. 24 shows that ATI-450 inhibits IL-17A production in TH17 cells.

FIG. 25 shows that ATI-450 inhibits IL-1β stimulated TNF-α, Il-6, andIL-8 production.

FIGS. 26A-26B show that ATI-450 inhibits both IL-1α biosynthesis inhuman peripheral blood mononuclear cells (FIG. 26A) and IL-1α activityin neutrophils (FIG. 26B).

FIG. 27 shows a PXRD of Compound (P)-I.

FIG. 28 shows TGA (top trace) and DSC (bottom trace) curves for Compound(P)-I.

DETAILED DESCRIPTION

Definitions

Before the present compositions and methods are described, it is to beunderstood that this invention is not limited to the particularprocesses, formulations, compositions, or methodologies described, asthese may vary. It is also to be understood that the terminology used inthe description is for the purpose of describing the particular versionsor embodiments only, and is not intended to limit the scope ofembodiments herein which will be limited only by the appended claims.Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art. Although any methods and materials similar or equivalent tothose described herein can be used in the practice or testing ofembodiments herein, the preferred methods, devices, and materials arenow described. All publications mentioned herein are incorporated byreference in their entirety. Nothing herein is to be construed as anadmission that embodiments herein are not entitled to antedate suchdisclosure by virtue of prior invention.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to a “MK2 inhibitor” is areference to one or more MK2 inhibitors and equivalents thereof known tothose skilled in the art, and so forth.

The transitional term “comprising,” which is synonymous with“including,” “containing,” or “characterized by,” is inclusive oropen-ended and does not exclude additional, non-recited elements ormethod steps. By contrast, the transitional phrase “consisting of”excludes any element, step, or ingredient not specified in the claim.The transitional phrase “consisting essentially of” limits the scope ofa claim to the specified materials or steps “and those that do notmaterially affect the basic and novel characteristic(s)” of the claimedinvention. The compositions and methods of the present disclosure cancomprise, consist essentially of, or consist of, the components or stepsdisclosed.

As used herein, the term “about” means plus or minus 10% of thenumerical value of the number with which it is being used. Therefore,about 50% means in the range of 45%-55%.

“Administering” when used in conjunction with a therapeutic means toadminister a therapeutic to a patient whereby the therapeutic positivelyimpacts the tissue to which it is targeted. Thus, as used herein, theterm “administering”, when used in conjunction with a MK2 inhibitorcompound, can include, but is not limited to, providing a MK2 inhibitorcompound into or onto the target tissue; providing a MK2 inhibitorcompound systemically to a patient by, e.g., oral administration wherebythe therapeutic reaches the target tissue.

As used herein, the term “a derivative thereof” refers to a saltthereof, a pharmaceutically acceptable salt thereof, an ester thereof, afree acid form thereof, a free base form thereof, a solvate thereof, adeuterated derivative thereof, a hydrate thereof, an N-oxide thereof, aclathrate thereof, a prodrug thereof, a polymorph thereof, astereoisomer thereof, a geometric isomer thereof, a tautomer thereof, amixture of tautomers thereof, an enantiomer thereof, a diastereomerthereof, a racemate thereof, a mixture of stereoisomers thereof, anisotope thereof (e.g., tritium, deuterium), or a combination thereof.

The term “substantially free” as used herein, alone or in combination,refers to the absence of isomers within the limits of detection ofanalytical methods such as nuclear magnetic resonance (NMR), gaschromatography/mass spectroscopy (GC/MS), high performance liquidchromatography (HPLC), or liquid chromatography/mass spectroscopy(LC/MS).

The term “condition” as used herein is intended to be generallysynonymous, and is used interchangeably with, the terms “disorder,”“syndrome,” and “disease”, in that all reflect an abnormal condition ofthe human or animal body or of one of its parts that impairs normalfunctioning, is typically manifested by distinguishing signs andsymptoms, and causes the human or animal to have a reduced duration orquality of life.

The term “combination therapy” means the administration of two or moretherapeutic agents to treat a therapeutic condition or disorderdescribed in the present disclosure. Such administration encompassesco-administration of these therapeutic agents in a substantiallysimultaneous manner, such as in a single capsule having a fixed ratio ofactive ingredients or in multiple, separate capsules for each activeingredient. In addition, such administration also encompasses use ofeach type of therapeutic agent in a sequential manner. In either case,the treatment regimen will provide beneficial effects of the drugcombination in treating the conditions or disorders described herein.

“MK2 inhibitor” is used herein to refer to a compound that exhibits anIC₅₀ with respect to mitogen-activated protein kinase-activated proteinkinase 2 (“MK2”) activity of no more than about 100 μM and moretypically not more than about 50 μM, as measured in the MK2 enzymeassays. IC₅₀ is the concentration of inhibitor which reduces theactivity of an enzyme (e.g., MK2) to half-maximal level. Compoundsdisclosed herein have been discovered to exhibit inhibition against MK2.In some embodiments, the compounds will exhibit an IC₅₀ with respect toMK2 of no more than about 1 nM. In some embodiments, the compounds willexhibit an IC₅₀ with respect to MK2 of no more than about 1 μM. In someembodiments, the compounds will exhibit an IC₅₀ with respect to MK2 ofabout 1 μM to about 50 μM. In certain embodiments, compounds willexhibit an IC₅₀ with respect to MK2 of no more than about 10 μM; infurther embodiments, compounds will exhibit an IC₅₀ with respect to MK2of no more than about 5 μM; in yet further embodiments, compounds willexhibit an IC₅₀ with respect to MK2 of not more than about 1 μM; in yetfurther embodiments, compounds will exhibit an IC₅₀ with respect to MK2of not more than about 300 nM, as measured in the MK2 assay describedherein.

As used herein, the term “pharmaceutically acceptable salt” refers to asalt prepared from a base or acid which is acceptable for administrationto a patient, such as a mammal. The term “pharmaceutically acceptablesalts” embraces salts commonly used to form alkali metal salts and toform addition salts of free acids or free bases. The nature of the saltis not critical, provided that it is pharmaceutically-acceptable. Suchsalts can be derived from pharmaceutically-acceptable inorganic ororganic bases and from pharmaceutically-acceptable inorganic or organicacids.

Suitable pharmaceutically acceptable acid addition salts of thecompounds of embodiments herein may be prepared from an inorganic acidor an organic acid. All of these salts may be prepared by conventionalmeans from the corresponding compound of embodiments herein by treating,e.g., the compound with the appropriate acid or base.

Pharmaceutically acceptable acids include both inorganic acids, forexample hydrochloric, hydrobromic, hydroiodic, nitric, carbonic,sulfuric, phosphoric and diphosphoric acid; and organic acids, forexample formic, acetic, trifluoroacetic, propionic, succinic, glycolic,embonic (pamoic), methanesulfonic, ethanesulfonic,2-hydroxyethanesulfonic, pantothenic, benzenesulfonic, toluenesulfonic,sulfanilic, mesylic, cyclohexylaminosulfonic, stearic, algenic,β-hydroxybutyric, malonic, galactic, galacturonic, citric, fumaric,gluconic, glutamic, lactic, maleic, malic, mandelic, mucic, ascorbic,oxalic, pantothenic, succinic, tartaric, benzoic, acetic, xinafoic(1-hydroxy-2-naphthoic acid), napadisilic (1,5-naphthalenedisulfonicacid) and the like.

Salts derived from pharmaceutically-acceptable inorganic bases suitablefor the formulations as described herein include aluminum, ammonium,calcium, copper, ferric, ferrous, lithium, magnesium, manganic,manganous, potassium, sodium, zinc and the like. Salts derived frompharmaceutically-acceptable organic bases include salts of primary,secondary and tertiary amines, including alkyl amines, arylalkyl amines,heterocyclyl amines, cyclic amines, naturally-occurring amines and thelike, such as arginine, betaine, caffeine, choline, chloroprocaine,diethanolamine, N-methylglucamine, N,N′-dibenzylethylenediamine,diethylamine, 2-diethylaminoethanol, 2-dimethylaminoethanol,ethanolamine, ethylenediamine, N-ethylmorpholine, N-ethylpiperidine,glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine and the like.

Other preferred salts according to embodiments herein are quaternaryammonium compounds wherein an equivalent of an anion (X−) is associatedwith the positive charge of the N atom. X− may be an anion of variousmineral acids (e.g., chloride, bromide, iodide, sulfate, nitrate,phosphate), or an anion of an organic acid (e.g., acetate, maleate,fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate,trifluoroacetate, methanesulfonate, p-toluenesulfonate). X⁻ ispreferably an anion selected from chloride, bromide, iodide, sulfate,nitrate, acetate, maleate, oxalate, succinate or trifluoroacetate. Morepreferably X⁻ is chloride, bromide, trifluoroacetate ormethanesulfonate.

The compounds of embodiments herein may exist in both non-solvated andsolvated forms. The term solvate is used herein to describe a molecularcomplex comprising a compound of embodiments herein and an amount of oneor more pharmaceutically acceptable solvent molecules. The term hydrateis employed when said solvent is water. Examples of solvate formsinclude, but are not limited to, compounds of embodiments herein inassociation with water, acetone, dichloromethane, 2-propanol, ethanol,methanol, dimethyl sulfoxide (DMSO), ethyl acetate, acetic acid,ethanolamine, or mixtures thereof. It is specifically contemplated thatin embodiments herein one solvent molecule can be associated with onemolecule of the compounds of embodiments herein, such as a hydrate.

In some embodiments herein one solvent molecule can be associated withone molecule of the compound described herein, such as a hydrate. Insome embodiments, more than one solvent molecule may be associated withone molecule of the compound described herein, such as a dihydrate.Additionally, in some embodiments herein less than one solvent moleculemay be associated with one molecule of the compound described herein,such as a hemihydrate. Furthermore, solvates of embodiments herein arecontemplated as solvates of the compound described herein that retainthe biological effectiveness of the non-solvate form of the compounds.

Embodiments herein also includes isotopically-labeled compounds ofembodiments herein, wherein one or more atoms is replaced by an atomhaving the same atomic number, but an atomic mass or mass numberdifferent from the atomic mass or mass number usually found in nature.Examples of isotopes suitable for inclusion in the compounds ofembodiments herein include isotopes of hydrogen, such as ²H and ³Hcarbon, such as ¹¹C, ¹³C and ¹⁴C, chlorine, such as ³¹Cl, fluorine, suchas ¹⁸F, iodine, such as ¹²³I and ¹²⁵I, nitrogen, such as ¹³N and 15N,oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O, phosphorus, such as ³²P, and sulfur,such as ³⁵S. Certain isotopically-labeled compounds of embodimentsherein, e.g., those incorporating a radioactive isotope, are useful indrug and/or substrate tissue distribution studies. The radioactiveisotopes tritium, ³H, and carbon-14, ¹⁴C, are particularly useful forthis purpose in view of their ease of incorporation and ready means ofdetection. Substitution with heavier isotopes such as deuterium, ²H, mayafford certain therapeutic advantages resulting from greater metabolicstability, e.g., increased in vivo half-life or reduced dosagerequirements, and hence may be preferred in some circumstances.Substitution with positron emitting isotopes, such as ¹¹C, ¹⁸F, ¹⁵O and¹³N, can be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy.

Isotopically-labeled compounds of embodiments herein can generally beprepared by conventional techniques known to those skilled in the art orby processes analogous to those described herein, using an appropriateisotopically-labeled reagent in place of the non-labeled reagentotherwise employed.

Preferred isotopically-labeled compounds include deuterated derivativesof the compounds of embodiments herein. As used herein, the termdeuterated derivative embraces compounds of embodiments herein where ina particular position at least one hydrogen atom is replaced bydeuterium. Deuterium (D or ²H) is a stable isotope of hydrogen which ispresent at a natural abundance of 0.015 molar %.

Hydrogen deuterium exchange (deuterium incorporation) is a chemicalreaction in which a covalently bonded hydrogen atom is replaced by adeuterium atom. Said exchange (incorporation) reaction can be total orpartial.

Typically, a deuterated derivative of a compound of embodiments hereinhas an isotopic enrichment factor (ratio between the isotopic abundanceand the natural abundance of that isotope (the percentage ofincorporation of deuterium at a given position in a molecule in theplace of hydrogen) for each deuterium present at a site designated as apotential site of deuteration on the compound of at least 3500 (52.5%deuterium incorporation).

In some embodiments, the isotopic enrichment factor is at least 5000(75% deuterium). In some embodiments, the isotopic enrichment factor isat least 6333.3 (95% deuterium incorporation). In some embodiments, theisotopic enrichment factor is at least 6633.3 (99.5% deuteriumincorporation). It is understood that the isotopic enrichment factor ofeach deuterium present at a site designated as a site of deuteration isindependent from the other deuteration sites.

The term “subject” as used herein and interchangeably with “patient”,includes, but is not limited to, humans and non-human vertebrates suchas wild, domestic, and farm animals. In certain embodiments, the subjectdescribed herein is an animal. In certain embodiments, the subject is amammal. In certain embodiments, the subject is a human. In certainembodiments, the subject is a non-human animal. In certain embodiments,the subject is a non-human mammal. In certain embodiments, the subjectis a domesticated animal, such as a dog, cat, cow, pig, horse, sheep, orgoat. In certain embodiments, the subject is a companion animal such asa dog or cat. In certain embodiments, the subject is a livestock animalsuch as a cow, pig, horse, sheep, or goat. In another embodiment, thesubject is a research animal such as a rodent, dog, or non-humanprimate. In certain embodiments, the subject is a non-human transgenicanimal such as a transgenic mouse or transgenic pig.

The phrase “therapeutically effective” is intended to qualify the amountof active ingredients used in the treatment of a disease or disorder oron the effecting of a clinical endpoint.

The term “therapeutically acceptable” refers to those compounds, and aderivative thereof, which are suitable for use in contact with thetissues of patients without undue toxicity, irritation, and allergicresponse, are commensurate with a reasonable benefit/risk ratio, and areeffective for their intended use.

The terms “treat,” “treated,” “treating”, or “treatment” as used hereinrefers to both therapeutic treatment and prophylactic or preventativemeasures, wherein the object is to prevent or slow down (lessen) anundesired physiological condition, disorder or disease, or to obtainbeneficial or desired clinical results. For the purposes of thisinvention, beneficial or desired clinical results include, but are notlimited to, alleviation of symptoms; diminishment of the extent of thecondition, disorder or disease; stabilization (i.e., not worsening) ofthe state of the condition, disorder or disease; delay in onset orslowing of the progression of the condition, disorder or disease;amelioration of the condition, disorder or disease state; and remission(whether partial or total, whether induction of or maintenance of),whether detectable or undetectable, or enhancement or improvement of thecondition, disorder or disease. Treatment includes eliciting aclinically significant response without excessive levels of sideeffects. Treatment also includes prolonging survival as compared toexpected survival if not receiving treatment. Treatment may also bepreemptive in nature, i.e., it may include prevention of disease.Prevention of a disease may involve complete protection from disease,for example as in the case of prevention of infection with a pathogen,or may involve prevention of disease progression. For example,prevention of a disease may not mean complete foreclosure of any effectrelated to the diseases at any level, but instead may mean prevention ofthe symptoms of a disease to a clinically significant or detectablelevel. Prevention of diseases may also mean prevention of progression ofa disease to a later stage of the disease and prolonging disease-freesurvival as compared to disease-free survival if not receiving treatmentand prolonging disease-free survival as compared to disease-freesurvival if not receiving treatment.

Embodiments herein are directed to oral pharmaceutical compositions thatinhibit MK2 activity and methods of treatment that involve administeringto a subject in need thereof an oral dose of the MK2 inhibitor compound.Some embodiments include methods for the treatment of diseases in asubject in need thereof that comprise orally administering the MK2inhibitor compound described herein.

The oral compositions disclosed herein possess a specific MK2 inhibitorwhich prevents p38 MAP Kinase mediated inflammatory signaling, and thus,can be used in the treatment or prophylaxis of a disease or condition inwhich p38 MAP Kinase inflammatory signaling plays an active role. Thus,embodiments provide oral pharmaceutical compositions comprising the MK2inhibitor disclosed herein together with a pharmaceutically acceptablecarrier, as well as methods for using the compounds and compositions.Certain embodiments provide methods for inhibiting p38 MAP kinaseinflammatory signaling using compounds of embodiments herein. Otherembodiments provide methods for treating a p38 MAP Kinase-mediateddisorder in a patient in need of such treatment, comprisingadministering to said patient a therapeutically effective amount of aMK2 inhibitor compound or composition comprising the same according tothe present disclosure. Also provided is the use of the specific MK2inhibitor disclosed herein for use in the manufacture of a medicamentfor the treatment of a disease or condition ameliorated by theinhibition of p38 MAP Kinase.

Also provided are embodiments wherein any embodiment described hereinmay be combined with any one or more of these embodiments, provided thecombination is not mutually exclusive.

Oral Compositions

Embodiments herein are directed to pharmaceutical compositionsformulated for oral administration (“oral pharmaceutical composition”),comprising about 5 mg to about 300 mg of Compound I as shown below

or a derivative thereof, and a pharmaceutically acceptable carrier.

Compound I is also referred to herein by its chemical name, i.e.,3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-2′-(2-(2-hydroxypropan-2-yl)pyrimidin-4-yl)-5′,6-dimethyl-2H-[1,4′-bipyridin]-2-one.

Compound I may be prepared according to the methods described in U.S.Pat. No. 9,115,089, which is hereby incorporated by reference in itsentirety. Compound I may also be obtained from Aclaris Therapeutics,Inc. (640 Lee Road, Suite 200, Wayne, Pa. 19087, USA).

There are two atropisomers of3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-2′-(2-(2-hydroxypropan-2-yl)pyrimidin-4-yl)-5′,6-dimethyl-2H-[1,4′-bipyridin]-2-one,which are depicted below as Compound (P)-I((P)-3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-2′-(2-(2-hydroxypropan-2-yl)pyrimidin-4-yl)-5′,6-dimethyl-2H-[1,4′-bipyridin]-2-one)and Compound (M)-I((M)-3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-2′-(2-(2-hydroxypropan-2-yl)pyrimidin-4-yl)-5′,6-dimethyl-2H-[1,4′-bipyridin]-2-one).

The term “atropisomerism” refers to a type of isomerism resulting fromhindered rotation around a single bond due to steric strain of thesubstituents. This phenomenon creates stereoisomers which display axialchirality. Atropisomers may be separated (resolved) via supercriticalfluid chromatography using a mobile phase of carbon dioxide andethanol/methanol. Chiral resolution of the P and M atropisomers of3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-2′-(2-(2-hydroxypropan-2-yl)pyrimidin-4-yl)-5′,6-dimethyl-2H-[1,4′-bipyridin]-2-oneis described in the Examples herein.

As used throughout this disclosure, recitation of “Compound I”encompasses atropisomer compounds (P)-I and (M)-I as depicted above inany molar ratio from 4:1 ((P)-I:(M)-I) to 999:1 and also includesembodiments where Compound (P)-I is substantially free from Compound(M)-I. Compounds (P)-I and (M)-I can be in any form (e.g., free base,crystalline form, etc.) as described herein.

In any embodiment, Compound I of the oral composition as disclosedherein comprises Compound (P)-I and Compound (M)-I in a molar ratio ofabout 4:1 ((P)-I:(M)-I) to about 999:1. In any embodiment, the molarratio of (P)-I to (M)-I is about 4.3:1, about 4.6:1, about 4.9:1, about5.25:1, about 5.7:1, about 6.1:1, about 6.7:1, about 7.3:1, about 8.1:1,about 9:1, about 10:1, about 11.5:1, about 13.3:1, about 15.7:1, about19:1, about 24:1, about 32.3:1, about 49:1, about 91:1, about 110.1:1,about 124:1, about 141.9:1, about 165.7:1, about 199:1, about 249:1,about 332.3:1, about 399:1, about 499:1, and about 999:1. In a preferredembodiment, the molar ratio of (P)-I to (M)-I is about 399:1.

Said another way, in any embodiment, Compound I of the oral compositionas disclosed herein comprises at least 80 mol % of Compound (P)-I. Inany embodiment the oral composition as disclosed herein comprises atleast 81 mol % of Compound (P)-I, at least 82 mol % of Compound (P)-I,at least 83 mol % of Compound (P)-I, at least 84 mol % of Compound(P)-I, at least 85 mol % of Compound (P)-I, at least 86 mol % ofCompound (P)-I, at least 87 mol % of Compound (P)-I, at least 88 mol %of Compound (P)-I, at least 89 mol % of Compound (P)-I, at least 90 mol% of Compound (P)-I, at least 91 mol % of Compound (P)-I, at least 92mol % of Compound (P)-I, at least 93 mol % of Compound (P)-I, at least94 mol % of Compound (P)-I, at least 95 mol % of Compound (P)-I, atleast 96 mol % of Compound (P)-I, at least 97 mol % of Compound (P)-I,at least 98 mol % of Compound (P)-I, at least 99 mol % of Compound(P)-I, at least 99.1 mol % of Compound (P)-I, at least 99.2 mol % ofCompound (P)-I, at least 99.3 mol % of Compound (P)-I, at least 99.4 mol% of Compound (P)-I, at least 99.5 mol % of Compound (P)-I, at least99.6 mol % of Compound (P)-I, at least 99.7 mol % of Compound (P)-I, atleast 99.8 mol % of Compound (P)-I, at least 99.9 mol % of Compound(P)-I. In a preferred embodiment the oral composition as disclosedherein comprises at least 99.75 mol % of Compound (P)-I. In anyembodiment, Compound I of the oral composition as disclosed hereincomprises Compound (P)-I substantially free from Compound (M)-I.

In any embodiment, the oral pharmaceutical composition disclosed hereincomprises 10 mg of Compound I. In any embodiment, the oralpharmaceutical composition disclosed herein comprises 40 mg of CompoundI. In any embodiment, the oral pharmaceutical composition disclosedherein comprises 50 mg of Compound I. In any embodiment, the oralpharmaceutical composition disclosed herein comprises 60 mg of CompoundI. In any embodiment, the oral pharmaceutical composition disclosedherein comprises 80 mg of Compound I. In any embodiment, the oralpharmaceutical composition disclosed herein comprises 100 mg of CompoundI. In any embodiment, the oral pharmaceutical composition disclosedherein comprises 120 mg of Compound I. In any embodiment, the oralpharmaceutical composition disclosed herein comprises 160 mg of CompoundI. In any embodiment, the oral pharmaceutical composition disclosedherein comprises 200 mg of Compound I. In any embodiment, the oralpharmaceutical composition disclosed herein comprises 240 mg of CompoundI.

In any embodiment, the oral pharmaceutical compositions described hereincomprise Compound I in an amount of about 5 mg to about 200 mg. In anyembodiment, Compound I is present in the pharmaceutical composition asdescribed herein in an amount of about 5 mg to about 300 mg, about 7.5mg to about 300 mg, about 10 mg to about 300 mg, about 12.5 mg to about300 mg, about 15 mg to about 300 mg, about 17.5 mg to about 300 mg,about 20 mg to about 300 mg, about 22.5 mg to about 300 mg, about 25 mgto about 300 mg, about 27.5 mg to about 300 mg, about 30 mg to about 300mg, about 32.5 mg to about 300 mg, about 35 mg to about 300 mg, about37.5 mg to about 300 mg, about 40 mg to about 300 mg, about 42.5 mg toabout 300 mg, about 45 mg to about 300 mg, about 47.5 mg to about 300mg, about 50 mg to about 300 mg, about 50 mg to about 290 mg, about 50mg to about 280 mg, about 50 mg to about 270 mg, about 50 mg to about260 mg, about 50 mg to about 250 mg, about 50 mg to about 240 mg, about50 mg to about 230 mg, about 50 mg to about 220 mg, about 50 mg to about210 mg, about 50 mg to about 200 mg about 50 mg to about 190 mg, about50 mg to about 180 mg, about 50 mg to about 170 mg, about 50 mg to about160 mg, about 50 mg to about 150 mg, about 50 mg to about 140 mg, about50 mg to about 130 mg, about 50 mg to about 120 mg, about 50 mg to about110 mg, about 50 mg to about 100 mg, about 50 mg to about 90 mg, about50 mg to about 80 mg, about 50 mg to about 70 mg, about 50 mg to about60 mg, about 40 mg to about 50 mg, about 30 mg to about 60 mg, about 20mg to about 70 mg, about 15 mg to about 80 mg, about 10 mg to about 90mg, about 5 mg to about 100 mg, or any amount in between. In a preferredembodiment, the oral pharmaceutical compositions described hereincomprise Compound I in an amount of about 50 mg to about 240 mg.

In any embodiment, Compound I is present in the pharmaceuticalcomposition as described herein in an amount of about 5 mg, 7.5 mg, 10mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5mg, 80 mg, 82.5 mg, 85 mg, 87.5 mg, 90 mg, 92.5 mg, 95 mg, 97.5 mg, 100mg, 105 mg, 107.5 mg, 110 mg, 112.5 mg, 115 mg, 117.5 mg, 120 mg, 122.5mg, 125 mg, 127.5 mg, 130 mg, 132.5 mg, 135 mg, 137.5 mg, 140 mg, 142.5mg, 145 mg, 147.5 mg, 150 mg, 152.5 mg, 155 mg, 157.5 mg, 160 mg, 162.5mg, 165 mg, 167.5 mg, 170 mg, 172.5 mg, 175 mg, 177.5 mg, 180 mg, 182.5mg, 185 mg, 187.5 mg, 190 mg, 192.5 mg, 195 mg, 197.5 mg, 200 mg, 200mg, 205 mg, 207.5 mg, 210 mg, 212.5 mg, 215 mg, 217.5 mg, 220 mg, 222.5mg, 225 mg, 227.5 mg, 230 mg, 232.5 mg, 235 mg, 237.5 mg, 240 mg, 242.5mg, 245 mg, 247.5 mg, 250 mg, 252.5 mg, 255 mg, 257.5 mg, 260 mg, 262.5mg, 265 mg, 267.5 mg, 270 mg, 272.5 mg, 275 mg, 277.5 mg, 280 mg, 282.5mg, 285 mg, 287.5 mg, 290 mg, 292.5 mg, 295 mg, 297.5 mg, or 300 mg. Inpreferred embodiments, Compound I is present in the pharmaceuticalcomposition as described herein in an amount of 50 mg, 80 mg, 100 mg,120 mg, 160 mg, or 240 mg.

In any embodiment, Compound I of the oral composition as disclosedherein comprises a free base. In any embodiment, Compound I of the oralcomposition as disclosed herein comprises a pharmaceutically acceptablesalt.

In any embodiment, Compound I of the oral composition comprises Compound(P)-I and Compound (M)-I as disclosed herein in the free base form. Inany embodiment, Compound I of the oral composition comprises Compound(P)-I and Compound (M)-I as disclosed herein in form of pharmaceuticallyacceptable salts.

In any embodiment, the pharmaceutically acceptable salt is an acidaddition salt. Suitable acid addition salts include those formed withboth organic and inorganic acids. Pharmaceutically acceptable acidsinclude both inorganic acids, for example hydrochloric, hydrobromic,hydroiodic, nitric, carbonic, sulfuric, phosphoric and diphosphoricacid; and organic acids, for example formic, acetic, trifluoroacetic,propionic, succinic, glycolic, embonic (pamoic), methanesulfonic,ethanesulfonic, 2-hydroxyethanesulfonic, pantothenic, benzenesulfonic,toluenesulfonic, sulfanilic, mesylic, cyclohexylaminosulfonic, stearic,algenic, 3-hydroxybutyric, malonic, galactic, galacturonic, citric,fumaric, gluconic, glutamic, lactic, maleic, malic, mandelic, mucic,ascorbic, oxalic, pantothenic, succinic, tartaric, benzoic, acetic,xinafoic (1-hydroxy-2-naphthoic acid), napadisilic(1,5-naphthalenedisulfonic acid) and the like.

In any embodiment, the pharmaceutically acceptable salt is a basicaddition salt. Basic addition salts can be prepared during the finalisolation and purification of the compounds by reacting a carboxy groupwith a suitable base such as the hydroxide, carbonate, or bicarbonate ofa metal cation or with ammonia or an organic primary, secondary, ortertiary amine. The cations of therapeutically acceptable salts includelithium, sodium, potassium, calcium, magnesium, and aluminum, as well asnontoxic quaternary amine cations such as ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, diethylamine, ethylamine, tributylamine, pyridine,N,N-dimethylaniline, N-methylpiperidine, N-methylmorpholine,dicyclohexylamine, procaine, dibenzylamine, N,N-dibenzylphenethylamine,1-ephenamine, and N,N′-dibenzylethylenediamine. Other representativeorganic amines useful for the formation of base addition salts includeethylenediamine, ethanolamine, diethanolamine, piperidine, andpiperazine.

In any embodiment, Compound I of the oral compositions is a non-solvatedform or in a solvated form. In any embodiment herein one solventmolecule can be associated with one molecule of Compound I describedherein, such as a hydrate. In some embodiments, more than one solventmolecule may be associated with one molecule of Compound I as describedherein, such as a dihydrate. Additionally, in some embodiments hereinless than one solvent molecule may be associated with one molecule ofCompound I described herein, such as a hemihydrate. Furthermore,solvates of embodiments herein are contemplated as solvates of CompoundI as described herein that retain the biological effectiveness of thenon-solvate form of Compound I.

In any embodiment, Compound 1 of the oral composition is a deuteratedderivative. As used herein, the term deuterated derivative embracescompounds of embodiments herein where in a particular position at leastone hydrogen atom is replaced by deuterium. Deuterium (D or ²H) is astable isotope of hydrogen which is present at a natural abundance of0.015 molar %.

In any embodiment, Compound I of the oral composition as disclosedherein comprises Compound (P)-I (free base) in a crystalline form. Inany embodiment, the crystalline form of Compound (P)-I is crystallineForm A as disclosed and characterized herein.

For example, the crystalline form A of Compound (P)-I of the oralcomposition may be characterized by its PXRD pattern. Thus, in anyembodiment, the crystalline Form A of Compound (P)-I is characterized byan PXRD pattern having a peak expressed in degrees 2θ at about 9.78±0.2.In any embodiment, the crystalline Form A of Compound (P)-I ischaracterized by an PXRD pattern having peaks expressed in degrees 2θ at9.78±0.2 and 15.51±0.2. In any embodiment, the crystalline Form A ofCompound (P)-I is characterized by an PXRD pattern having peaksexpressed in degrees 2θ at 9.78±0.2, 15.51±0.2, 19.6±0.2, and 25.92±0.2.In any embodiment, the crystalline Form A of Compound (P)-I ischaracterized by an PXRD pattern having peaks expressed in degrees 2θ at9.78±0.2, 15.34±0.2, 15.51±0.2, 19.6±0.2, 20.57±0.2, 21.01±0.2,25.92±0.2, 29.05±0.2, and 29.48±0.2. In any embodiment, the crystallineForm A of Compound (P)-I is characterized by an PXRD pattern of FIG. 27.

The crystalline Form A of Compound (P)-I of the oral compositiondisclosed herein may be additionally or alternatively characterized bythermogravimetric analysis (TGA). Samples of crystalline Form A of (P)-Iyielded a TGA curve revealing that, in the sample analyzed, negligibleweight loss was observed. Weight loss (0.7%) is observed between 25° C.and 256° C. by TGA for freebase crystalline Form A, suggesting thatcrystalline form A of (P)-I is substantially anhydrous.

The crystalline form A of (P)-I may additionally or alternatively becharacterized by differential scanning calorimetry (DSC). Thus, in anyembodiment, the crystalline Form A of Compound (P)-I is characterized bya DSC plot comprising an initial endothermic melting event with an onsettemperature of about 188° C., followed by an exothermicrecrystallization event at about 196° C., with a final sharp endothermicmelting event at about 254° C.

In any embodiment, the oral composition of the present disclosurecomprises Compound I as disclosed herein formulated by admixture with apharmaceutically acceptable carrier or excipient. In certainembodiments, the pharmaceutical compositions include the therapeuticallyeffective amount of Compound I and a physiologically acceptable diluentor carrier. In certain embodiments, the pharmaceutical compositionfurther includes one or more additional therapeutic components and/oradjuvants.

In any embodiment, the oral compositions disclosed herein may furthercomprise pharmaceutically acceptable diluents, fillers, disintegrants,binders, lubricants, surfactants, hydrophobic vehicles, water solublevehicles, emulsifiers, buffers, humectants, moisturizers, solubilizers,preservatives and the like. The means and methods for preparation andadministration are known in the art and an artisan can refer to variouspharmacologic references for guidance. For example, Banker, G. S., &Rhodes, C. T. (2002). Modern pharmaceutics. New York: Marcel Dekker.;and Goodman, L. S., Brunton, L. L., Chabner, B., & Knollmann, B. C.(2011). Goodman & Gilman's pharmacological basis of therapeutics. NewYork: McGraw-Hill. can be consulted.

The oral pharmaceutical compositions as disclosed herein can beformulated readily by combining Compound I with pharmaceuticallyacceptable carriers well known in the art. Such carriers enable thecompounds of embodiments herein to be formulated as nanoparticles,nanoparticle suspension, tablets, troches, pills, dragees, capsules,powders, liquids, gels, syrups, slurries, suspensions and the like, fororal ingestion by a subject to be treated. Oral compositions can also beprepared using a fluid carrier for use as a mouthwash, wherein thecompound in the fluid carrier is applied orally and swished andexpectorated or swallowed. Pharmaceutical preparations for oraladministration can be obtained by adding a solid excipient, optionallygrinding the resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients include, but are not limited to,fillers such as sugars, including, but not limited to, lactose, sucrose,mannitol, and sorbitol; cellulose preparations such as, but not limitedto, maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropyl methylcellulose (HPMC),sodium carboxymethylcellulose (CMC), and polyvinylpyrrolidone (PVP). Ifdesired, disintegrating agents can be added, such as, but not limitedto, the cross-linked PVP, agar, or alginic acid or a salt thereof suchas sodium alginate.

Dragee cores can be provided with suitable coatings. For this purpose,concentrated sugar solutions can be used, which can optionally containgum arabic, talc, PVP, carbopol gel, polyethylene glycol (PEG), and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments can be added to the tablets ordragee coatings for identification or to characterize differentcombinations of active compound doses.

Pharmaceutical preparations which can be used orally include, but arenot limited to, push-fit capsules made of gelatin, as well as soft,sealed capsules made of gelatin and a plasticizer, such as glycerol orsorbitol. The push-fit capsules can contain the active ingredient in anadmixture with one or more fillers (e.g., lactose), one or more binders(e.g., starches), and/or one or more lubricants (e.g., talc or magnesiumstearate) and, optionally, one or more stabilizers. In soft capsules,the active compound can be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid PEG. In addition,stabilizers can be added. All compositions for oral administrationshould be in dosages (e.g., about 5 mg to about 300 mg) suitable forsuch administration.

In some embodiments, the oral compositions may take the form of, e.g.,lozenges, tablets or capsules prepared by conventional means withpharmaceutically acceptable excipients such as binding agents (e.g.,pregelatinized maize starch, PVP or HPMC); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulfate). The tablets may be coated by methods well known in theart with, e.g., sugars, films or enteric coatings. Additionally, thepharmaceutical compositions containing Compound I as disclosed hereincan be in any form suitable for oral use, including, e.g., troches,lozenges, aqueous or oily suspensions, dispersible powders or granules,emulsions, hard or soft capsules, or syrups or elixirs. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions and suchcompositions may contain one or more agents selected from the groupconsisting of sweetening agents, flavoring agents, coloring agents andpreserving agents in order to provide pharmaceutically elegant andpalatable preparations.

In one embodiment, the oral pharmaceutical composition as disclosedherein is a tablet. Tablets may contain Compound I in admixture withnon-toxic pharmaceutically acceptable excipients which are suitable forthe manufacture of tablets. These excipients may be e.g., inertdiluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents(e.g., corn starch, or alginic acid); binding agents (for examplestarch, gelatin or acacia); and lubricating agents (for examplemagnesium stearate, stearic acid or talc). The tablets may be uncoatedor they may be coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction over a longer period. In some embodiments, the tablet isformulated for immediate release. In some embodiments, the tablet isformulated for controlled release. For example, a time delay materialsuch as glyceryl monostearate or glyceryl distearate may be employed.They may also be coated by the techniques described in the U.S. Pat.Nos. 4,256,108; 4,166,452; and 4,265,874 to form osmotic therapeutictablets for control release. The pharmaceutical compositions describedherein may also be in the form of oil-in-water emulsions.

In some embodiments, the oral composition comprises Compound I and abuffer. In some embodiments, the buffer may be selected from the groupconsisting of citric acid monohydrate, sodium phosphate, water, and acombination thereof. In some embodiments, the oral composition comprisesCompound I and a stabilizer. In some embodiments, the stabilizer isselected from a group consisting of povidone, sodium benzoate, water,sodium lauryl sulfate, and a combination thereof. In some embodiments,the oral composition further includes a buffer, an acid, sodiumbenzoate, sodium phosphate, citric acid, or a combination thereof. Insome embodiments, the oral composition comprises Compound I and astabilizer and a buffer. In some embodiments, the oral compositionfurther comprises a lubricant, a pH modifier, a binder, a diluent, agranulating agent, a glidant, a disintegrant, a filler, a sorbent, ananti-adherent, a coloring agent, a compression aid, a coating material,a sweetener, a preservative, an antioxidant, or a combination thereof.In some embodiments, Compound I is in a therapeutically effective amount(e.g., about 5 mg to about 200 mg). In some embodiments, the oralcomposition is a suspension, tablet, capsule, nanoparticle powder,nanoparticle suspension, cachet, pellet, pill, powder, granules, or acombination thereof.

In some embodiments, the lubricant may be selected from the groupconsisting of stearic acid or its salts (e.g., magnesium stearate,calcium stearate), sodium lauryl sulfate, PEG, mineral oil, sodiumbenzoate, glyceryl palmitostearate, glyceryl behenate, sodium stearylfumarate, and a combination thereof.

In some embodiments, the pH modifier may be an acid (e.g., hydrochloricacid, acetic acid, citric acid, phosphoric acid, sulfuric acid, or acombination thereof).

In some embodiments, the binder may be selected from the groupconsisting of a natural or synthetic polymer (e.g., starches, sugars,sugar alcohols, or cellulose derivatives) such as gelatin, glucose,lactose, sorbitol, xylitol, maltitol, methyl cellulose, microcrystallinecellulose (MCC), ethyl cellulose, HPMC, hydroxypropyl cellulose (HPC),starch, PVP, PEG, sodium alginate, CMC, and a combination thereof.

In some embodiments, the compression aid may be selected from the groupconsisting of silicified microcrystalline cellulose, microcrystallinecellulose, a physical mixture of MCC-colloidal silicon dioxide, and acombination thereof.

In some embodiments, the disintegrant may be selected from the groupconsisting of starch, cellulose derivatives and alginates, PVP,croscarmellose sodium, sodium starch glycolate, and a combinationthereof.

In some embodiments, the filler may be selected from the groupconsisting of lactose, sucrose, glucose, mannitol, sorbitol, calciumcarbonate, magnesium stearate, plant cellulose, dibasic calciumphosphate, dibasic sodium phosphate, vegetable fats and oils, and acombination thereof.

In some embodiments, the diluent may be selected from the groupconsisting of sugar compounds (e.g., sucrose, lactose, dextrin, glucose,sorbitol, or the like), inorganic compounds (e.g., silicates, calciumsalts, or magnesium salts), sodium chloride, potassium chloride, and acombination thereof.

In some embodiments, the preservative may be selected from the groupconsisting of an antioxidant (e.g., vitamin A, vitamin E, vitamin C,retinyl palmitate, and selenium), an amino acid (e.g., cysteine, ormethionine), citric acid, sodium citrate, a synthetic preservative(e.g., a paraben such as methyl paraben or propyl paraben), and acombination thereof.

In some embodiments, the glidant may be selected from the groupconsisting of colloidal anhydrous silicon and other silica compounds,such as fumed silica, magnesium carbonate, colloidal silicon dioxide(AEROSIL®), cornstarch, talc, and a combination thereof.

In some embodiments the oral composition comprising Compound I is acapsule. In some embodiments, the capsule comprises an inner coatingmade from a high fat emulsion. In some embodiments the capsule comprisesa high fat coating that is either on the inside or the outside of thecapsule. In some embodiments the capsule comprises HPMC. In someembodiments, the capsule may be a HPMC capsule. In some embodiments, thecapsule may be enteric coated. In some embodiments, the capsule may be asilica capsule, such as silica sold under the trade name SYLOID®. Insome embodiments, the capsule comprises cyclodextrin. In someembodiments, the capsule may be a cyclodextrin complex enteric capsule.

In some embodiments the oral formulation comprising Compound I is atablet. In some embodiments the tablet contains Compound I in the formof nanoparticles. In some embodiments, the tablet may be coated. In someembodiments, the tablet may be coated with an enteric coating. In someembodiments, the tablet may be coated with a coating selected from asugar coating, film coating, organic film coating, aqueous film coating,pan coating, dip coating, electrostatic coating, compression coating,plasticizer dry coating, heat dry coating, electrostatic dry coating, orthe like. Some ingredients used for coating may include aqueous acrylicenteric system such as that sold under the trade name ACRYL-EZE®, filmcoating system sold under the trade name OPADRY®, HPMC, methylhydroxyethyl cellulose, ethylcellulose, povidone, cellulose acetatephthalate, acrylate polymers (such as those sold under the trade nameEUDRAGIT® L & EUDRAGIT® S), HPMC phthalate, or a combination thereof.

In some embodiments the oral composition comprises Compound I in theform of a nanoparticle suspension (nanosuspension). In some embodiments,the nanosuspension comprises Compound I, a stabilizer, and a buffer. Insome embodiments, the nanosuspension may further comprise a pH modifier.In some embodiments, the pH modifier may be selected from a groupconsisting of hydrochloric acid, acetic acid, citric acid, phosphoricacid, sulfuric acid, and a combination thereof. In some embodiments, thepH modifier may be hydrochloric acid. In some embodiments, thehydrochloric acid may be 1.0N hydrochloric acid. In some embodiments,the stabilizer may be selected from the group consisting of povidone,sodium lauryl sulfate, sodium benzoate, WFI quality water such as thatsold under the tradename HYCLONE™, or a combination thereof. In someembodiments, the buffer solution may include WFI quality water, sodiumphosphate (dibasic, 7-hydrate, crystal), citric acid monohydrate, or acombination thereof.

The nanoparticle suspension may be manufactured by suspending particlesof the active in the excipients, reducing particles to the desiredparticle size using grinding media in a mill, and then diluting thesuspension to the final volume. In some embodiments, the grinding mediaused may be selected from ceramic, agate, silicon nitride, sinteredcorundum, zirconia, stainless steel, chrome steel, Cr—Ni steel, tungstencarbide, glass (yttrium-stabilized), cross-linked polystyrene resins,plastic polyamide, pearls, or a combination thereof. In someembodiments, the mill may be a stationary agitated vessel or arecirculating mill.

A tablet may be manufactured by spraying the nanosuspension (above) ontosucrose to form a spray granulate intermediate, granulating the spraygranulate intermediate with excipients to form a final granulation, andcompressing the final granulation to form a tablet. The sucrose could beany sugar, including, e.g., glucose, fructose, maltose, galactose,lactose, or the like. In some embodiments, the excipients for the tabletformulation may include lactose monohydrate, PVP, silicifiedmicrocrystalline cellulose (e.g., sold under the trade name PROSOLV®SMCC HD 90), magnesium stearate, or a combination thereof. In someembodiments, the tablet comprises about 5 mg to about 300 mg of CompoundI.

In some embodiments the oral composition as described herein is a drypowder. In some embodiments, the dry powder may be encapsulated or madeinto a suspension. In some embodiments, the suspension may be ananoparticle suspension or a milled suspension.

In some embodiments, liquid preparations for oral administration maytake the form of, e.g., elixirs, solutions, syrups or suspensions, orthey may be presented as a dry product for constitution with water orother suitable vehicle before use. Such liquid preparations may beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (e.g., sorbitol syrup, cellulosederivatives or hydrogenated edible fats); emulsifying agents (e.g.,lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oilyesters, ethyl alcohol, CREMOPHORE® or fractionated vegetable oils); andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid). The preparations may also contain buffer salts, preservatives,flavoring, coloring and sweetening agents as appropriate.

Pharmaceutical compositions of the compounds also can comprise suitablesolid or gel phase carriers or excipients. Examples of such carriers orexcipients include but are not limited to calcium carbonate, calciumphosphate, various sugars, starches, cellulose derivatives, gelatin, andpolymers (e.g., PEG).

The compositions can further include one or more additionalpharmaceutical agents such as an anti-inflammatory drug, ananti-atherosclerotic drug, an immunosuppressive drug, animmunomodulatory drug, a cytostatic drug, an angiogenesis inhibitor, akinase inhibitor, a cytokine blocker, and an inhibitor of cell adhesionmolecules.

Methods of Use

Another aspect of the present disclosure relates a method for treatingan inflammatory condition in a subject in need thereof. This methodcomprises administering, to a human subject having an inflammatorycondition, an oral dose of 5 mg/day to 300 mg/day of Compound I, i.e.,Compound (P)-I and (M)-I in any molar ratio as described supra and inany form (e.g., free form, crystalline form) as described supra. Aparticularly useful oral dose of Compound I for use in the methodsdescribed herein comprises an oral dose of 100 mg/day to 240 mg/day ofCompound I comprising greater than 80 mol % of Compound (P)-I.

Suitable inflammatory conditions that can be treated in accordance withthe methods disclosed herein include any inflammatory conditioninvolving the p38 MAP kinase-mediated inflammatory signaling. In someembodiments the inflammatory condition is a chronic inflammatorycondition. In some embodiments, the inflammatory condition is an acuteinflammatory condition. In some embodiments, the inflammatory conditionis an autoinflammatory condition. In some embodiments, the inflammatorycondition is an autoimmune condition. In some embodiments, theinflammatory condition is inflammasomopathy.

For example, in any embodiment, the methods and compositions disclosedherein are suitable for treating chronic or acute inflammatory orautoimmune gastrointestinal disorders, inflammatory or autoimmune skindisorders, neuroinflammatory disorders, inflammatory heart disease,inflammatory lung diseases, inflammatory myopathies, inflammatory bonedisorders or diseases, periodic fever syndromes, as well as pain orpruritus associated with any aforementioned disease.

Compound I, in any embodiment, also be used to treat scarring/fibroticdiseases or disorders and various types of cancers and hyperproliferative disorders.

For example, in any embodiment, the methods and compositions disclosedherein are suitable for treating inflammatory arthritis, such asrheumatoid arthritis (RA), spondyloarthritis such as ankylosingspondylitis, psoriatic arthritis, reactive arthritis and Reiter'ssyndrome, juvenile rheumatoid arthritis (JIA), systemic-onset juvenilerheumatoid arthritis, idiopathic arthritis (JIA) (including systemic(SJIA)), and gout; cryopyrin-associated autoinflammatory syndromes(CAPS), including Muckle-Wells syndrome (MWS), neonatal-onsetmultisystem inflammatory disease (NOMID), and familial coldautoinflammatory syndrome (FCAS); chronic obstructive pulmonary diseases(COPD), including emphysema, chronic bronchitis, and asthma (allergicand non-allergic); inflammatory skin conditions, including, but notlimited to hidradenitis suppurativa (HS), psoriasis, such as plaquepsoriasis, pyoderma gangrenosum, IL-17 associated skin condition,pruritus; colitis from an inflammatory bowel disease (IBD) such asCrohn's disease or ulcerative colitis and inflammatory boweldisease-associated arthritis; pericarditis, including acutepericarditis, recurrent pericarditis, and chronic pericarditis;pulmonary inflammation or fibrosis, including idiopathic pulmonaryfibrosis and interstitial lung disease; metastatic breast cancer, andpancreatic cancer.

In any embodiment, the methods and compositions disclosed herein aresuitable for treating Familial Mediterranean Fever (FMF); tumor necrosisfactor receptor-associated periodic syndrome (TRAPS); adult-onsetStill's disease; pyoderma gangrenosum; bone-resorption disorders (suchas those associated with cancer (e.g., breast cancer)); metastaticmelanoma; Castleman disease; and chronic atypical neutrophilicdermatosis with lipodystrophy (CANDLE).

In some embodiments, the condition that is treated in accordance withthe methods described here is pruritus, which may be associated with anyother condition, for example, pruritus associated with hidradenitissuppurativa, pruritus associated with inflammation, pruritus associatedwith rheumatoid arthritis, pruritus associated with psoriasis, andpruritus associated with TH17-associated inflammation.

In any embodiment, the methods and compositions disclosed herein aresuitable for treating Lyme disease; cytokine release syndrome (CRS);acute respiratory distress syndrome (ARDS); chronic or acute bronchitis;epidermolysis bullosa (EB); bullous pemphigoid; juveniledermatomyositis; inflammatory vitiligo (including marginal); pemphigusvulgaris; enterocolitis; polymyositis; myositis, bone cancer; lungcancer; inflammatory bone disorders such as chronic recurrent multiosteomyelitis (CRMO), Synovitis, acne, pustulosis, hyperostosis, andosteitis (SAPHO) syndrome, Majeed syndrome, deficiency of interleukin-1receptor antagonist (DIRA) and cherubism; bone resorption (such as isassociated with an autoimmune disease); neuroinflammatory diseases suchas Alzheimer's disease (AD), Parkinson's disease (PD), multiplesclerosis (MS), acute disseminated encephalomyelitis (ADEM), acute opticneuritis (AON), transverse myelitis, and neuromyelitis optical (NMO);Behcet's disease; endotoxic shock (e.g., toxic shock syndrome (TSS) andother systemic gram-negative bacterial infections); enthesitis;polyarteritis nodosa (PAN); chronic pain; polymyalgia rheumatica;chronic allograft rejection; Sjogren's syndrome; and Schnitzler'ssyndrome (SchS).

In any embodiment, the condition that is treated in accordance with themethods described here is arthritis, such as inflammatory arthritis,rheumatoid arthritis (RA), spondyloarthritis such as ankylosingspondylitis, psoriatic arthritis, reactive arthritis and Reiter'ssyndrome, juvenile rheumatoid arthritis (JIA), systemic-onset juvenilerheumatoid arthritis, idiopathic arthritis (JIA) (including systemic(SJIA)), gout, and inflammatory bowel disease-associated arthritis.

In any embodiment, the condition to be treated is rheumatoid arthritis,ankylosing spondylitis, or psoriatic arthritis.

In any embodiment, the condition that is treated is an inflammatory skincondition, such as, hidradenitis suppurativa, psoriasis, such as plaquepsoriasis, pyoderma gangrenosum, an IL-17 related inflammatory skincondition, and a IL-1α related inflammatory skin condition.

In any embodiment, the condition to be treated is cryopyrin associatedperiodic syndrome (CAPS), including MWS, NOMIDS, and FCAS.

In any embodiment, the condition to be treated is irritable boweldisease, including colitis, Crohn's disease and ulcerative colitis.

In any embodiment, the condition to be treated is cytokine releasesyndrome, for example, CAR-T cell induced cytokine release syndrome, andacute respiratory distress syndrome.

In any embodiment, the condition to be treated is cancer, including, butnot limited to, metastatic breast cancer, pancreatic cancer, colorectalcancer and lung cancer.

In any embodiment, the condition to be treated is chronic obstructivepulmonary diseases (COPD), including emphysema, chronic bronchitis, andasthma (allergic and non-allergic).

In any embodiment, the condition to be treated is pulmonary fibrosis,including idiopathic pulmonary fibrosis and interstitial lung disease.

Other conditions that may be treated in accordance with the methods andcompositions disclosed herein include inflammatory and/or autoimmuneconditions such as allergic and non-allergic asthma, pancreatitis,autoimmune encephalomyelitis, autoimmune myositis, giant-cell arteritis,episcleritis, glomerulonephritis, Hashimoto's thyroiditis, keratitis,lupus nephritis, myocarditis, enteritis, neutrophilic eccrinehidradentitis, nonalcoholic steatohepatitis, periodontitis,polychondritis, primary sclerosing cholangitis, schleritis, sinusitis,small vessel vasculitis, large vessel vasculitis, Takayasu's arteritis,atopic dermatitis, autoimmune and inflammatory hepatitis, autoimmuneatrophic gastritis, autoimmune orchitis, bronchiolitis, bronchioligisobliterans, carditis, chortitis, esophagitis, hepatitis C, opticneurotis, uveitis, tinea capitis, acne vulgaris, and hypophysitis,lupus, myasthenia gravis, pernicious anemia, type I diabetes, Addison'sdisease, Chagas disease, peripheral neuropathy, hypoxia or ischemiainduced inflammation, autoimmune nephropathy, cicatricial pemphigoid,Goodpasture's Disease, Graves' disease, histiocytoid neutrophilicdermatosis, hypereosinophilic syndrome, stimulator of interferongenes-associated vasculopathy with onset in infancy (SAVI),Churg-Strauss syndrome, Guillain-Barret syndrome, immune-mediatedglomerulonephritis, linear IgA disease, sarcoidosis, sympatheticophthalmia, and type 3 hypersensitivity reaction disease.

Other conditions that may be treated in accordance with the methods andcompositions disclosed herein include any condition where MK2 inhibitionis therapeutically beneficial, such as cancer (e.g., head/neck cancer,bladder cancer, intestinal cancer, non-small cell lung cancer,astrocytoma, small cell lung cancer, colon cancer, colorectal cancer,esophageal cancer, fibrotic cancers, hepatic cancers, leukemia, kidneycancer, larynx cancer, multiple myeloma, Merkel cell carcinoma, mouth orpharynx cancer, nerve cancer, non-melanoma skin cancer, ovarian cancer,prostate cancer, renal cancer, seminoma, squamous cell carcinoma,stomach cancer, schwannoma, teratocarcinoma, testicular cancer,osteosarcoma, rhabdomyosarcoma, Wegener's granulomatosis,keratocanthoma, Kaposi's sarcoma, glioblastoma, glioma, t-cell lymphoma,throat cancer, thyroid cancer, thyroid follicular cancer, and uterinecancer), fibrotic conditions (e.g., atrial fibrosis, cardiac fibrosis,cystic fibrosis, endomyocardial fibrosis, hepatic fibrosis, idiopathicmyelofibrosis (IMF), mediastinal fibrosis, myelofibrosis, nephrogenicsystemic fibrosis, renal fibrosis, retroperitoneal fibrosis,fibroadenomas, fibromyalgia, fibrosarcomas, fibrosclerosis, fibroids,fibroma, fibrosing alopecia, glomerulosclerosis, juvenile scleroderma,membranous glomerulopathy, schleroderma, and cardioa-allograftvasculopathy), and gastrointestinal fibrosis), retinopathy, cirrhosis,and keratopathy.

As described supra, the oral composition suitable for administration inaccordance with the methods described herein comprises a therapeuticallyeffective amount (e.g., about 50 mg/day to about 300 mg/day) of CompoundI (i.e., in a molar ratio of Compound (P)-I:Compound (M)-I) of 4:1 to999:1) or an oral pharmaceutical composition comprising the same.

The specific dose level for any particular patient will depend upon avariety of factors including the activity of the specific compoundemployed, the age, body weight, general health, sex, diet, time ofadministration, rate of excretion, drug combination, the precisedisorder being treated, and the severity of the indication or conditionbeing treated.

In some embodiments, administration of Compound I (i.e., in a molarratio of Compound (P)-I:Compound (M)-I) of 4:1 to 999:1) or an oralpharmaceutical composition comprising the same is effective to cause atleast partial remission of the symptoms that characterize the disease.In some embodiments, administration of one or more of the presentlydisclosed oral compositions of embodiments herein is effective to causeat least full remission of the symptoms that characterize the disease.

In any embodiment, administration of a composition comprising Compound Ias described herein is effective to cause inhibition of p38 MAPkinase-mediated pro-inflammatory, but not anti-inflammatory signaling.In some embodiments, administration of one or more of the presentlydisclosed oral compositions of embodiments herein is effective to causeinhibition of MK2 inflammatory signaling. Inhibition of p38 MAP kinaseand MK2 mediated inflammatory signaling can be measured or assessed byin vivo serum levels of one or more inflammatory cytokines, including,but not limited to TNF-α, IL-1β, IL-6, IL-8, IFNγ, IL-17, IL-18, IL-1α,and MIP1β. Thus in any embodiment, administration of a compositioncomprising Compound I, in particular, a composition comprising at least80 mol %, at least 90 mol %, at least 95 mol %, or at least 99 mol % ofCompound (P)-I at the dosages described herein (e.g., 100 mg/day, 160mg/day, or 240 mg/day) reduces the in vivo serum levels of one or morecytokines selected from TNF-α, IL-1β, IL-6, IL-8, IFNγ, IL-17, IL-18,IL-1α, and MIP1β in a subject as compared to the in vivo serum levels ofthe corresponding cytokines prior to treatment of the subject with thecomposition comprising Compound I.

In one embodiment, the inflammatory condition to be treated inaccordance with the methods and compositions comprising Compound Idescribed herein is arthritis, in particular moderate to severerheumatoid arthritis. In some embodiments, treatment of a subject havingmoderate to severe rheumatoid arthritis in accordance with the methodsand compositions comprising Compound I as described herein, alone or incombination with methotrexate, is effective to inhibit progression ofjoint damage, improve synovitis, or both as assessed by magneticresonance imaging (MRI).

In some embodiments, treatment of a subject having moderate to severerheumatoid arthritis in accordance with the methods and compositionscomprising Compound I as described herein, alone or in combination withmethotrexate, is effective to reduce high sensitivity C-reactive protein(hsCRP) levels in the subject. In some embodiments, hsCRP is reducedrelative to the baseline level (i.e., the hsCRP level prior totreatment) by at least 10%, by at least 20%, by at least 30%, by atleast 40%, or by greater than 40%. Preferably, treatment with theCompound I reduced hsCRP levels by greater than 40% relative tobaseline.

In some embodiments, treatment of a subject having moderate to severerheumatoid arthritis in accordance with the methods and compositionscomprising Compound I as described herein, alone or in combination withmethotrexate, is effective to reduce the subject's Disease ActivityScore for 28 Joint Count (DAS28). The DAS28 consists of a compositescore of the following variables: tender joint count, swollen jointcount, CRP, and Patient's Global Assessment of Disease Activity score.Interpretation of the DAS28 (CRP) disease activity measure is on a scaleof 0 to 9.4, where: <2.6 is considered remission, ≥2.6 to <3.2 isconsidered low/minimal, ≥3.2 to ≤5.1 is considered moderate, and >5.1 isconsidered high/severe (Anderson et al., “Rheumatoid arthritis diseaseactivity measures: American College of Rheumatology recommendations foruse in clinical practice,” Arthritis Care Res (Hoboken) 64(5):640-7(2012), which is hereby incorporated by reference in its entirety).Thus, in some embodiments, treatment of moderate to severe rheumatoidarthritis in accordance with the methods and compositions comprisingCompound I as described herein is effective to reduce the DAS28(CRP)score to ≤3.2 over the course of treatment as an indicator of lowdisease activity of remission. In some embodiments, treatment of severerheumatoid arthritis in accordance with the methods and compositionscomprising Compound I as described herein is effective to reduce theDAS28(CRP) score to <2.6 over the course of treatment as an indicator ofremission.

In some embodiments, treatment of a subject having moderate to severerheumatoid arthritis in accordance with the methods and compositionscomprising Compound I as described herein, alone or in combination withmethotrexate, is effective to achieve at least 20% improvement in theswollen and tender joints counts and an improvement of least 20% in atleast 3 of the following 5 measures: (i) Patient's Global Assessment ofDisease Activity (VAS), (ii) Patient's Assessment of Arthritis Pain(VAS), (iii) Patient's Assessment of Physical Function/Health AssessmentQuestionnaire—Disability Index (HAQ-DI), (iv) Physician's GlobalAssessment of Disease Activity (VAS), (v) Acute phase reactant asmeasured by hsCRP. In some embodiments, treatment with compositionscomprising Compounds I as describe herein, alone or in combination withmethotrexate, is effective to achieve at least 50% improvement in theswollen and tender joints counts and an improvement of least 50% in atleast 3 of the 5 above noted measures. In some embodiments, treatmentwith compositions comprising Compound I as describe herein, alone or incombination with methotrexate, is effective to achieve at least 70%improvement in the swollen and tender joints counts and an improvementof least 70% in at least 3 of the 5 above noted measures.

In some embodiments, treatment of a subject having moderate to severerheumatoid arthritis in accordance with the methods and compositionscomprising Compound I as described herein, alone or in combination withmethotrexate, is effective to significantly reduce predosing levels ofone or more inflammatory cytokines. In some embodiments, treatment of asubject having severe rheumatoid arthritis with a composition comprisingCompound I, alone or in combination with methotrexate, is effective tosignificantly reduce predosing levels of one or more of TNF-α, IL-6,IL-8, IL-1β, and MIP1β. In any embodiment, the aforementioned cytokinelevels are reduced by least 10%, at least 15%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, or greaterthan 75% following administration of a composition comprising Compound Ias disclosed in the Examples herein.

In some embodiments, treatment of a subject having moderate to severeactive psoriatic arthritis in accordance with the methods andcompositions comprising Compound I as described herein, alone or incombination with methotrexate, is effective to achieve at least a 20%reduction (improvement) compared with baseline in tender joint count(TJC), swollen joint count (SJC), and an improvement of least 20% in atleast at least 3 of the 5 remaining ACR core set measures: (i) patient'sassessment of pain, (ii) patient's global assessment of disease activity(PtGA); (iii) physician's global assessment of disease activity (PhGA),(iv) Health Assessment Questionnaire—Disability Index (HAQ-DI), and (iv)high sensitivity C-reactive protein (hsCRP).

In some embodiments, treatment of a subject having moderate to severeactive psoriatic arthritis in accordance with the methods andcompositions comprising Compound I as described herein, alone or incombination with methotrexate, is effective to achieve a staticInvestigator Global Assessment (sIGA) of Psoriasis of 0 or 1 and atleast a 2-point improvement from baseline (pre-treatment) sIGA levels.

In some embodiments, treatment of a subject having moderate to severeactive psoriatic arthritis in accordance with the methods andcompositions comprising Compound I as described herein, alone or incombination with methotrexate, is effective to achieve a psoriasis areaseverity index (PAST) 75 response (for participants with at least a 3%BSA psoriasis as baseline).

In some embodiments, treatment of a subject having moderate to severeactive psoriatic arthritis in accordance with the methods andcompositions comprising Compound I as described herein, alone or incombination with methotrexate, is effective to achieve minimal diseaseactivity (MDA), determined based on meeting 5 of 7 outcome measures: TJC≤1; SJC ≤1; PASI ≤1 or BSA-Ps ≤3%; Patient's Assessment of Pain NRS≤1.5; PtGA-Disease Activity NRS ≤2.0; HAQ-DI score ≤0.5; and tenderentheseal points ≤1.

In some embodiments, treatment of a subject having moderate to severeactive psoriatic arthritis in accordance with the methods andcompositions comprising Compound I as described herein, alone or incombination with methotrexate, is effective to significantly reducepredosing levels of one or more of TNF-α, IL-1β, IL-6, IL-8, IFNγ, IL17, IL-18, IL-1α and MIP1β. In any embodiment, the aforementionedcytokine levels are reduced by least 10%, at least 15%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, orgreater than 75% following administration of a composition comprisingCompound I.

In some embodiments, treatment of a subject having moderate to severeHidradenitis Suppurativa (HS) in accordance with the methods andcompositions comprising Compound I as described herein, alone or incombination with antibiotics, is effective to achieve a HidradenitisSuppurativa Clinical Response (HiSCR) during the course of treatment,where HiSCR is defined as at least a 50% reduction from baseline in thetotal abscess and inflammatory nodule (AN) count, with no increase inabscess or draining fistula counts.

In some embodiments, treatment of a subject having moderate to severeHidradenitis Suppurativa (HS) in accordance with the methods andcompositions comprising Compound I as described herein, alone or incombination with antibiotics, is effective to achieve at least 30%reduction from baseline (pre-treatment) level in Numerical Rating Scale(NRS30) in Patient's Global Assessment of Skin Pain (PGA Skin Pain)during the course of treatment.

In some embodiments, treatment of a subject having moderate to severeHidradenitis Suppurativa (HS) in accordance with the methods andcompositions comprising Compound I as described herein, alone or incombination with antibiotics, is effective to achieve at least 25%decrease in AN counts with a minimum increase of 2 relative to baselineover the treatment period.

In some embodiments, treatment of a subject having moderate to severeHidradenitis Suppurativa (HS) in accordance with the methods andcompositions comprising Compound I as described herein, alone or incombination with antibiotics, is effective to achieve at least one ormore of the following: increase from baseline in Dermatology LifeQuality Index (DLQI) with treatment (the DLQI is a 10-item validatedquestionnaire used to assess the impact of HS disease symptoms andtreatment on quality of life (QoL); decrease from baseline in HS-relatedswelling assessed based on the Hidradenitis Suppurativa SymptomAssessment (HSSA) with treatment (HSSA is a 9-item patient reportedoutcome (PRO) questionnaire developed to assess the symptoms of HS on a0 to 11-point NRS, where 0 represents no symptoms and 10 representsextreme symptom experience); decrease from baseline in HS-related odorassessed based on the HSSA over 12-weeks; change from baseline inHS-related worst drainage assessed based on the HSSA with treatment.

In some embodiments, treatment of a subject having moderate to severeHidradenitis Suppurativa (HS) in accordance with the methods andcompositions comprising Compound I as described herein, alone or incombination with antibiotics, is effective to achieve decrease frombaseline in one or more endogenous cytokine levels selected from TNF-αIL-1β, IL-6, IL-8, IFNγ, IL 17, IL-18, MIP1β, and IL-1α. In anyembodiment, the aforementioned cytokine levels are reduced by least 10%,at least 15%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, or greater than 75% following administration ofa composition comprising Compound I.

In some embodiments, treatment of a subject having aCryopyrin-associated Periodic Syndrome (CAPS) (e.g., Familial ColdAutoinflammatory Syndrome, Muckle-Wells Syndrome, or Neonatal OnsetMultisystem Inflammatory Disease) in accordance with the methods andcompositions comprising Compound I as described herein, is effective tocause disease remission as defined by one or both of (i) a PhysicianGlobal Assessment (PGA) score of absent or minimal, and (ii) a highsensitivity C-reactive protein (hsCRP) and serum amyloid A (SAA) valuewithin the normal range (≤10 mg/L) or within 30 percent of the baselinevalue.

In some embodiments, treatment of a subject having aCryopyrin-associated Periodic Syndrome (CAPS) (e.g., Familial ColdAutoinflammatory Syndrome, Muckle-Wells Syndrome, or Neonatal OnsetMultisystem Inflammatory Disease) in accordance with the methods andcompositions comprising Compound I as described herein, is effective tocause clinical remission as defined by a Physician Global Assessment(PGA) score of absent or minimal.

In some embodiments, treatment of a subject having aCryopyrin-associated Periodic Syndrome (CAPS) (e.g., Familial ColdAutoinflammatory Syndrome, Muckle-Wells Syndrome, or Neonatal OnsetMultisystem Inflammatory Disease) in accordance with the methods andcompositions comprising Compound I as described herein, is effective tomaintain a mean Key Symptom Score (KSS) of no more than 2 points higherthan baseline with treatment. KSS is derived from thepatient-administered daily health assessment form (DHAF), and is theaverage on a 0 to 10 scale (0=None, 10=Very Severe) of 5 separatescales—rash, feeling of fever and chills, joint pain, eye redness andpain, and fatigue.

In some embodiments, treatment of a subject having aCryopyrin-associated Periodic Syndrome (CAPS) (e.g., Familial ColdAutoinflammatory Syndrome, Muckle-Wells Syndrome, or Neonatal OnsetMultisystem Inflammatory Disease) in accordance with the methods andcompositions comprising Compound I as described herein, is effective tosignificantly reduce predosing levels of one or more of TNF-α, IL-6,IL-8, IL-10, and MIP1β. In any embodiment, the aforementioned cytokinelevels are reduced by least 10%, at least 15%, at least 20%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, or greaterthan 75% following administration of a composition comprising CompoundI.

In any embodiment, the oral compositions of Compound I described hereinmay be administered at a first dose to prevent progression, at a seconddose to induce remission, and/or a third dose to prevent the diseaseand/or maintain remission of the disease. Such doses may be the samedose, a lower dose, or a higher dose. The dose may be administered morefrequently, less frequently or at the same frequency. In someembodiments, the dose may be administered in combination with anothertherapy, a therapeutic, an adjuvant, or the like.

As described supra, subjects suitable for treatment in accordance withthe methods described herein include, humans and non-human vertebratessuch as wild, domestic, and farm animals. In some embodiments, thesubject described herein is an animal. In some embodiments, the subjectis a mammal. In come embodiments, the subject is a human. In someembodiments, the subject is a non-human animal. In some embodiments, thesubject is a non-human mammal.

Dosing Regimen

Compound I, i.e., Compound (P)-I and (M)-I in any molar ratio asdescribed supra and in any form (e.g., free form, crystalline form) asdescribed supra, or an oral pharmaceutical composition comprising thesame is administered in an amount effective to inhibit p38 MAPKinase-mediated inflammatory signaling in a subject in need thereof.Subjects in need of such therapy are disclosed above. In someembodiments, the subject is one having an inflammatory condition, e.g.,a chronic inflammatory condition, an acute inflammatory condition, animmune-inflammatory condition, an autoimmune condition, or aninflammasomopathy. In accordance with the methods disclosed herein, atherapeutically effective amount comprises 5 mg/day to 300 mg/day ofCompound I. The dosage to be administered to a particular subject willdepend on the characteristics of the subject being treated, e.g., theparticular subject treated, age, weight, health, types of concurrenttreatment, if any, and frequency of treatments, and can be easilydetermined by one of skill in the art (e.g., by the clinician).

In any embodiment, 10 mg/day of Compound I is administered to a subjecthaving a condition disclosed herein. In any embodiment, 40 mg/day ofCompound I is administered to a subject having a condition disclosedherein. In any embodiment, 50 mg/day of Compound I is administered to asubject having a condition disclosed herein. In any embodiment, 60mg/day of Compound I is administered to a subject having a conditiondisclosed herein. In any embodiment, 80 mg/day of Compound I isadministered to a subject having a condition disclosed herein. In anyembodiment, 100 mg/day of Compound I is administered to a subject havinga condition disclosed herein. In any embodiment, 120 mg/day of CompoundI is administered to a subject having a condition disclosed herein. Inany embodiment, 160 mg/day of Compound I is administered to a subjecthaving a condition disclosed herein. In any embodiment, 200 mg/day ofCompound I is administered to a subject having a condition disclosedherein. In any embodiment, 240 mg/day of Compound I is administered to asubject having a condition disclosed herein.

In any embodiment, the therapeutically effective amount of Compound I is5 mg/day to 300 mg/day, about 7.5 mg/day to about mg/day, about 10mg/day to about 300 mg/day, about 12.5 mg/day to about 300 mg/day, about15 mg/day to about 300 mg/day, about 17.5 mg/day to about 300 mg/day,about 20 mg/day to about 300 mg/day, about 22.5 mg/day to about 300mg/day, about 25 mg/day to about 300 mg/day, about 27.5 mg/day to about300 mg/day, about 30 mg/day to about 300 mg/day, about 32.5 mg/day toabout 300 mg/day, about 35 mg/day to about 300 mg/day, about 37.5 mg/dayto about 300 mg/day, about 40 mg/day to about 300 mg/day, about 42.5mg/day to about 300 mg/day, about 45 mg/day to about 300 mg/day, about47.5 mg/day to about 300 mg/day, about 50 mg/day to about 300 mg/day,about 50 mg/day to about 290 mg/day, about 50 mg/day to about 280mg/day, about 50 mg/day to about 270 mg/day, about 50 mg/day to about260 mg/day, about 50 mg/day to about 250 mg/day, about 50 mg/day toabout 240 mg/day, about 50 mg/day to about 230 mg/day, about 50 mg/dayto about 220 mg/day, about 50 mg/day to about 210 mg/day, about 50mg/day to about 200 mg/day, about 50 mg/day to about 190 mg/day, about50 mg/day to about 180 mg/day, about 50 mg/day to about 170 mg/day,about 50 mg/day to about 160 mg/day, about 50 mg/day to about 150mg/day, about 50 mg/day to about 140 mg/day, about 50 mg/day to about130 mg/day, about 50 mg/day to about 120 mg/day, about 50 mg/day toabout 110 mg/day, about 50 mg/day to about 100 mg/day, about 50 mg/dayto about 90 mg/day, about 50 mg/day to about 80 mg/day, about 50 mg/dayto about 70 mg/day, about 50 mg/day to about 60 mg/day, about 40 mg/dayto about 50 mg/day, about 30 mg/day to about 60 mg/day, about 20 mg/dayto about 70 mg/day, about 15 mg/day to about 80 mg/day, about 10 mg/dayto about 90 mg/day, about 5 mg/day to about 100 mg/day, or any amount inbetween. In a preferred embodiment the therapeutically effective amountof Compound I is about 100 mg/day to about 240 mg/day.

In some embodiments the therapeutically effective amount of Compound Icomprises 5 mg/day, 7.5 mg/day, 10 mg/day, 12.5 mg/day, 15 mg/day 17.5mg/day, 20 mg/day, 22.5 mg/day, 25 mg/day, 27.5 mg/day, 30 mg/day, 32.5mg/day, 35 mg/day, 37.5 mg/day, 40 mg/day, 42.5 mg/day, 45 mg/day, 47.5mg/day, 50 mg/day, 52.5 mg/day, 55 mg/day, 57.5 mg/day, 60 mg/day, 62.5mg/day, 65 mg/day, 67.5 mg/day, 70 mg/day, 72.5 mg/day, 75 mg/day, 77.5mg/day, 80 mg/day, 82.5 mg/day, 85 mg/day, 87.5 mg/day, 90 mg/day, 92.5mg/day, 95 mg/day, 97.5 mg/day, 100 mg/day, 105 mg/day, 107.5 mg/day,110 mg/day, 112.5 mg/day, 115 mg/day, 117.5 mg/day, 120 mg/day, 122.5mg/day, 125 mg/day, 127.5 mg/day, 130 mg/day, 132.5 mg/day, 135 mg/day,137.5 mg/day, 140 mg/day, 142.5 mg/day, 145 mg/day, 147.5 mg/day, 150mg/day, 152.5 mg/day, 155 mg/day 157.5 mg/day, 160 mg/day, 162.5 mg/day,165 mg/day, 167.5 mg/day, 170 mg/day, 172.5 mg/day, 175 mg/day, 177.5mg/day, 180 mg/day, 182.5 mg/day, 185 mg/day, 187.5 mg/day, 190 mg/day,192.5 mg/day, 195 mg/day, 197.5 mg/day, 200 mg/day, 205 mg/day, 207.5mg/day, 210 mg/day, 212.5 mg/day, 215 mg/day, 217.5 mg/day, 220 mg/day,222.5 mg/day, 225 mg/day, 227.5 mg/day, 230 mg/day, 232.5 mg/day, 235mg/day, 237.5 mg/day, 240 mg/day, 242.5 mg/day, 245 mg/day, 247.5mg/day, 250 mg/day, 252.5 mg/day, 255 mg/day 257.5 mg/day, 260 mg/day,262.5 mg/day, 265 mg/day, 267.5 mg/day, 270 mg/day, 272.5 mg/day, 275mg/day, 277.5 mg/day, 280 mg/day, 282.5 mg/day, 285 mg/day, 287.5mg/day, 290 mg/day, 292.5 mg/day, 295 mg/day, 297.5 mg/day, 300 mg/day.In preferred embodiments, the therapeutically effective amount ofCompound I comprises 100 mg/day, 160 mg/day, or 240 mg/day.

In any embodiment, the oral dose of Compound I that is administered tothe subject in accordance with the methods disclosed herein comprisesCompound (P)-I and Compound (M)-I in a molar ratio of about 4:1((P)-I:(M)-I). In any embodiment, the molar ratio of (P)-I to (M)-I isabout 4.3:1, about 4.6:1, about 4.9:1, about 5.25:1, about 5.7:1, about6.1:1, about 6.7:1, about 7.3:1, about 8.1:1, about 9:1, about 10:1,about 11.5:1, about 13.3:1, about 15.7:1, about 19:1, about 24:1, about32.3:1, about 49:1, about 91:1, about 110.1:1, about 124:1, about141.9:1, about 165.7:1, about 199:1, about 249:1, about 332.3:1, about399:1, about 499:1, and about 999:1. A particularly useful oral dose ofCompound I comprises a molar ratio of (P)-I to (M)-I is about 399:1.

In any embodiment, the oral dose of Compound I that is administered tothe subject in accordance with the methods disclosed herein comprises atleast 80 mol % of Compound (P)-I. In any embodiment the oral compositionas disclosed herein comprises at least 81 mol % of Compound (P)-I, atleast 82 mol % of Compound (P)-I, at least 83 mol % of Compound (P)-I,at least 84 mol % of Compound (P)-I, at least 85 mol % of Compound(P)-I, at least 86 mol % of Compound (P)-I, at least 87 mol % ofCompound (P)-I, at least 88 mol % of Compound (P)-I, at least 89 mol %of Compound (P)-I, at least 90 mol % of Compound (P)-I, at least 91 mol% of Compound (P)-I, at least 92 mol % of Compound (P)-I, at least 93mol % of Compound (P)-I, at least 94 mol % of Compound (P)-I, at least95 mol % of Compound (P)-I, at least 96 mol % of Compound (P)-I, atleast 97 mol % of Compound (P)-I, at least 98 mol % of Compound (P)-I,at least 99 mol % of Compound (P)-I, at least 99.1 mol % of Compound(P)-I, at least 99.2 mol % of Compound (P)-I, at least 99.3 mol % ofCompound (P)-I, at least 99.4 mol % of Compound (P)-I, at least 99.5 mol% of Compound (P)-I, at least 99.6 mol % of Compound (P)-I, at least99.7 mol % of Compound (P)-I, at least 99.8 mol % of Compound (P)-I, atleast 99.9 mol % of Compound (P)-I. A particularly useful oral dose ofCompound I comprises at least 99.75 mol % of Compound (P)-I.

In any embodiment, the oral dose of Compound I that is administered tothe subject in accordance with the methods disclosed herein comprisesCompound (P)-I substantially free from Compound (M)-I.

In any embodiment, the oral dose of Compound I that is administered tothe subject in accordance with the methods disclosed herein comprises afree base. In any embodiment, the oral dose of Compound I that isadministered to the subject in accordance with the methods disclosedherein comprises a pharmaceutically acceptable salt.

In any embodiment, the oral dose of Compound I that is administered tothe subject in accordance with the methods disclosed herein comprisesCompound (P)-I and Compound (M)-I as disclosed herein in the free baseform. In any embodiment, Compound I of the oral composition comprisesCompound (P)-I and Compound (M)-I as disclosed herein in form ofpharmaceutically acceptable salts.

In any embodiment, the oral dose of Compound I that is administered tothe subject in accordance with the methods disclosed herein comprisesCompound (P)-I (free base) in a crystalline form. In any embodiment, thecrystalline form of Compound (P)-I is crystalline Form A as disclosedand characterized herein.

For example, the crystalline form A of Compound (P)-I of the oralcomposition may be characterized by its PXRD pattern. Thus, in anyembodiment, the crystalline Form A of Compound (P)-I is characterized byan PXRD pattern having a peak expressed in degrees 2θ at about 9.78±0.2.In any embodiment, the crystalline Form A of Compound (P)-I ischaracterized by an PXRD pattern having peaks expressed in degrees 2θ at9.78±0.2 and 15.51±0.2. In any embodiment, the crystalline Form A ofCompound (P)-I is characterized by an PXRD pattern having peaksexpressed in degrees 2θ at 9.78±0.2, 15.51±0.2, 19.6±0.2, and 25.92±0.2.In any embodiment, the crystalline Form A of Compound (P)-I ischaracterized by an PXRD pattern having peaks expressed in degrees 2θ at9.78±0.2, 15.34±0.2, 15.51±0.2, 19.6±0.2, 20.57±0.2, 21.01±0.2,25.92±0.2, 29.05±0.2, and 29.48±0.2. In any embodiment, the crystallineForm A of Compound (P)-I is characterized by an PXRD pattern of FIG. 27.

The crystalline form A of (P)-I may additionally or alternatively becharacterized by differential scanning calorimetry (DSC). Thus, in anyembodiment, the crystalline Form A of Compound (P)-I is characterized bya DSC plot comprising an initial endothermic melting event with an onsettemperature of about 188° C., followed by an exothermicrecrystallization event at about 196° C., with a final sharp endothermicmelting event at about 254° C.

In some embodiments, treatment of a subject having an inflammatorycondition in accordance with the methods and compositions comprisingCompound I as described herein, is effective to achieve a maximum plasmaconcentration (C_(max)) of Compound I of 39±10.4 ng/mL with a mediant_(max) (time for Compound I to reach C_(max)) of 2.0 hours andtime-averaged concentration of Compound I circulating in the plasma(i.e., AUC_(0−t)) of 276±77.8 h*ng/mL.

In another embodiment, administration of a composition comprisingCompound I to a subject having an inflammatory condition is effective toachieve a C_(max) of 122.0±33.4 with a median t_(max) of 4.0 (range2.0-4.1) hours and AUC_(0−t) of 1074.0±243.5 h*ng/mL.

In another embodiment, administration of a composition comprisingCompound Ito a subject having an inflammatory condition is effective toachieve a C_(max) of 160.7±20.4 with a median t_(max) of 3.0 (range2.0-4.0) hours and AUC_(0−t) of 1430.0±254.0 h*ng/mL.

In another embodiment, administration of a composition comprisingCompound I to a subject having an inflammatory condition is effective toachieve a C_(max) of 426.0±110.6 with a median t_(max) of 2.0 (2.0-4.0)hours and AUC_(0−t) of 3489.8±475.7 h*ng/mL.

In another embodiment, administration of a composition comprisingCompound I to a subject having an inflammatory condition is effective toachieve a C_(max) of 51.8±15.8 with a median t_(max) of 2.0 (2.0-2.0)hours and AUC_(0−t) of 364.6±110.7 h*ng/mL.

In another embodiment, administration of a composition comprisingCompound I to a subject having an inflammatory condition is effective toachieve a C_(max) of 146.5±33.6 with a median t_(max) of 2.0 (1.0-4.0)hours and AUC_(0−t) of 1204.6±309.1 h*ng/mL

Preferably, administration of a composition comprising Compound I to asubject having an inflammatory condition is effective to achieve aC_(max) of 219.0±77.8 with a median t_(max) of 3.0 (1.0-4.0) hours andAUC_(0−t) of 2260.3±1074.7 h*ng/mL.

Preferably, administration of a composition comprising Compound I to asubject having an inflammatory condition is effective to achieve aC_(max) of 389±101 with a median t_(max) of 2.0 (2.0-3.0) hours andAUC_(0−t) of 4110±1170 h*ng/mL

Preferably, administration of a composition comprising Compound I to asubject having an inflammatory condition is effective to achieve aC_(max) of 417±123 with a median t_(max) of 2.0 (1.0-4.0) hours andAUC_(0−t) of 4270±2130 h*ng/mL

In any embodiment a subject having an inflammatory condition isadministered 5 mg/day to 300 mg/day of Compound I. In accordance withthis embodiment the subject has an inflammatory condition selected fromrheumatoid arthritis, hidradenitis suppurativa, gout, plaque psoriasis,psoriatic arthritis, ankylosing spondylitis, pericarditis, includingacute pericarditis, recurrent pericarditis, and chronic pericarditis,cryopyrin associated periodic syndrome (CAPS), including Muckle WellsSyndrome and familial cold autoinflammatory syndrome, pyodermagangrenosum, irritable bowel disease, including Crohn's disease andulcerative colitis, Stills disease, also referred to as juvenileidiopathic arthritis, atopic dermatitis, acute coronary syndrome, heartfailure, and cancer, including, but not limited to, breast cancer,pancreatic cancer, colorectal cancer and lung cancer.

In any embodiment, a subject having inflammatory arthritis, such asrheumatoid arthritis (RA), spondyloarthritis (such as ankylosingspondylitis and psoriatic arthritis), juvenile rheumatoid (JIA) oridiopathic arthritis (JIA) (including systemic (SJIA)), and gout;cryopyrin-associated autoinflammatory syndrome (CAPS), includingMuckle-Wells syndrome (MWS), neonatal-onset multisystem inflammatorydisease (NOMID), and familial cold autoinflammatory syndrome (FCAS);chronic obstructive pulmonary diseases (COPD), hidradenitis suppurativa(HS); psoriasis, such as plaque psoriasis; colitis from an inflammatorybowel disease (IBD) like Crohn's disease or ulcerative colitis;pericarditis; metastatic breast cancer, and pancreatic cancer isadministered an oral composition comprising 100 mg of Compound I thereofonce daily.

In any embodiment, a subject having inflammatory arthritis, such asrheumatoid arthritis (RA), spondyloarthritis (such as ankylosingspondylitis and psoriatic arthritis), juvenile rheumatoid (JIA) oridiopathic arthritis (JIA) (including systemic (SJIA)), and gout;cryopyrin-associated autoinflammatory syndrome (CAPS), includingMuckle-Wells syndrome (MWS), neonatal-onset multisystem inflammatorydisease (NOMID), and familial cold autoinflammatory syndrome (FCAS);chronic obstructive pulmonary diseases (COPD), hidradenitis suppurativa(HS); psoriasis, such as plaque psoriasis; colitis from an inflammatorybowel disease (IBD) like Crohn's disease or ulcerative colitis;pericarditis; metastatic breast cancer, and pancreatic cancer isadministered an oral composition comprising 160 mg of Compound I oncedaily.

In any embodiment, a subject having inflammatory arthritis, such asrheumatoid arthritis (RA), spondyloarthritis (such as ankylosingspondylitis and psoriatic arthritis), juvenile rheumatoid (JIA) oridiopathic arthritis (JIA) (including systemic (SJIA)), and gout;cryopyrin-associated autoinflammatory syndrome (CAPS), includingMuckle-Wells syndrome (MWS), neonatal-onset multisystem inflammatorydisease (NOMID), and familial cold autoinflammatory syndrome (FCAS);chronic obstructive pulmonary diseases (COPD), hidradenitis suppurativa(HS); psoriasis, such as plaque psoriasis; colitis from an inflammatorybowel disease (IBD) like Crohn's disease or ulcerative colitis;pericarditis; metastatic breast cancer, and pancreatic cancer isadministered an oral composition comprising 200 mg of Compound I oncedaily.

In any embodiment, a subject having inflammatory arthritis, such asrheumatoid arthritis (RA), spondyloarthritis (such as ankylosingspondylitis and psoriatic arthritis), juvenile rheumatoid (JIA) oridiopathic arthritis (JIA) (including systemic (SJIA)), and gout;cryopyrin-associated autoinflammatory syndrome (CAPS), includingMuckle-Wells syndrome (MWS), neonatal-onset multisystem inflammatorydisease (NOMID), and familial cold autoinflammatory syndrome (FCAS);chronic obstructive pulmonary diseases (COPD), hidradenitis suppurativa(HS); psoriasis, such as plaque psoriasis; colitis from an inflammatorybowel disease (IBD) like Crohn's disease or ulcerative colitis;pericarditis; metastatic breast cancer, and pancreatic cancer isadministered an oral composition comprising 240 mg of Compound I oncedaily.

In any embodiment, a subject having inflammatory arthritis, such asrheumatoid arthritis (RA), spondyloarthritis (such as ankylosingspondylitis and psoriatic arthritis), juvenile rheumatoid (JIA) oridiopathic arthritis (JIA) (including systemic (SJIA)), and gout;cryopyrin-associated autoinflammatory syndrome (CAPS), includingMuckle-Wells syndrome (MWS), neonatal-onset multisystem inflammatorydisease (NOMID), and familial cold autoinflammatory syndrome (FCAS);chronic obstructive pulmonary diseases (COPD), hidradenitis suppurativa(HS); psoriasis, such as plaque psoriasis; colitis from an inflammatorybowel disease (IBD) like Crohn's disease or ulcerative colitis;pericarditis; metastatic breast cancer, and pancreatic cancer isadministered an oral composition comprising 50 mg of Compound I twicedaily.

In any embodiment, a subject having inflammatory arthritis, such asrheumatoid arthritis (RA), spondyloarthritis (such as ankylosingspondylitis and psoriatic arthritis), juvenile rheumatoid (JIA) oridiopathic arthritis (JIA) (including systemic (SJIA)), and gout;cryopyrin-associated autoinflammatory syndrome (CAPS), includingMuckle-Wells syndrome (MWS), neonatal-onset multisystem inflammatorydisease (NOMID), and familial cold autoinflammatory syndrome (FCAS);chronic obstructive pulmonary diseases (COPD), hidradenitis suppurativa(HS); psoriasis, such as plaque psoriasis; colitis from an inflammatorybowel disease (IBD) like Crohn's disease or ulcerative colitis;pericarditis; metastatic breast cancer, and pancreatic cancer isadministered an oral composition comprising 80 mg of Compound I twicedaily.

In any embodiment, a subject having inflammatory arthritis, such asrheumatoid arthritis (RA), spondyloarthritis (such as ankylosingspondylitis and psoriatic arthritis), juvenile rheumatoid (JIA) oridiopathic arthritis (JIA) (including systemic (SJIA)), and gout;cryopyrin-associated autoinflammatory syndrome (CAPS), includingMuckle-Wells syndrome (MWS), neonatal-onset multisystem inflammatorydisease (NOMID), and familial cold autoinflammatory syndrome (FCAS);chronic obstructive pulmonary diseases (COPD), hidradenitis suppurativa(HS); psoriasis, such as plaque psoriasis; colitis from an inflammatorybowel disease (IBD) like Crohn's disease or ulcerative colitis;pericarditis; metastatic breast cancer, and pancreatic cancer isadministered an oral composition comprising 100 mg of Compound I twicedaily.

In any embodiment, a subject having inflammatory arthritis, such asrheumatoid arthritis (RA), spondyloarthritis (such as ankylosingspondylitis and psoriatic arthritis), juvenile rheumatoid (JIA) oridiopathic arthritis (JIA) (including systemic (SJIA)), and gout;cryopyrin-associated autoinflammatory syndrome (CAPS), includingMuckle-Wells syndrome (MWS), neonatal-onset multisystem inflammatorydisease (NOMID), and familial cold autoinflammatory syndrome (FCAS);chronic obstructive pulmonary diseases (COPD), hidradenitis suppurativa(HS); psoriasis, such as plaque psoriasis; colitis from an inflammatorybowel disease (IBD) like Crohn's disease or ulcerative colitis;pericarditis; metastatic breast cancer, and pancreatic cancer isadministered an oral composition comprising 120 mg of Compound I twicedaily.

In any embodiment, a subject having rheumatoid arthritis is administeredorally 50 mg/day to 300 mg/day of Compound I. In some embodiments, thesubject having rheumatoid arthritis is administered an oral compositioncomprising Compound I once a day. In some embodiments, the subjecthaving rheumatoid arthritis is administered a composition comprisingCompound I twice a day. In some embodiments, the subject havingrheumatoid arthritis is administered an oral composition comprising 10mg, 30 mg, 50 mg, 80 mg, 100 mg, 120 mg, 160 mg, 200 mg, or 240 mg ofCompound I once daily.

In any embodiment, the subject having rheumatoid arthritis isadministered an oral composition comprising 100 mg of Compound I oncedaily.

In any embodiment, the subject having rheumatoid arthritis isadministered an oral composition comprising 160 mg of Compound I oncedaily.

In any embodiment, the subject having rheumatoid arthritis isadministered an oral composition comprising 200 mg of Compound I oncedaily.

In any embodiment, the subject having rheumatoid arthritis isadministered an oral composition comprising 240 mg of Compound I oncedaily.

In any embodiment, the subject having rheumatoid arthritis isadministered an oral composition comprising 10 mg, 30 mg, 50 mg, 80 mg,100 mg, or 120 mg of Compound I twice daily.

In any embodiment, the subject having rheumatoid arthritis isadministered an oral composition comprising 50 mg of Compound I twicedaily.

In any embodiment, the subject having rheumatoid arthritis isadministered an oral composition comprising 80 mg of Compound I twicedaily.

In any embodiment, the subject having rheumatoid arthritis isadministered an oral composition comprising 100 mg of Compound I twicedaily.

In any embodiment, the subject having rheumatoid arthritis isadministered an oral composition comprising 120 mg of Compound I twicedaily.

The dosage administered is a therapeutically effective amount of thecomposition sufficient to result in amelioration of a symptom orsymptoms, and can vary depending upon known factors such as thepharmacodynamic characteristics of the active ingredient and its mode ofadministration; age, sex, health and weight of the recipient; nature andextent of symptoms; kind of concurrent treatment, frequency of treatmentand the effect desired.

In some embodiments, the oral composition comprising Compound I asdescribed herein can be administered to the subject once (e.g., as asingle dose or application). In some embodiments, the oral compositionof embodiments herein is administered at least once daily, such as atleast two, three or four times daily. In some embodiments, the oralcomposition of embodiments herein may be administered daily, twicedaily, three times daily, weekly, twice weekly, every two weeks, everythree weeks, monthly, as needed, or as otherwise directed by aphysician. The oral composition of embodiments herein may beadministered at any interval to achieve the therapeutically desiredeffect, e.g., induction or maintenance of remission, prevention orrelief of a symptom or symptoms. In some embodiments, the oralcomposition of embodiments herein may be administered to a subject for aperiod of 1, 2, 3, 4, 5, 6 days, about a week, about two weeks, aboutthree weeks, about four weeks, about five weeks, about six weeks, abouttwo months, about three months, about four months, about five months,about six months, or a range of any two of these values. In someembodiments, treatment may be continued for at least a week, a month, ayear, or as otherwise directed by a physician. In some embodiments,treatment may extend over multiple years, the duration of disease, orthe lifetime of the subject. In some embodiments, the oral compositionof embodiments herein can be administered once or twice daily to asubject in need thereof for a period of about two to about twenty-eightdays, or from about seven to about ten days. The oral composition ofembodiments herein can also be administered once, twice, or three timesdaily to a subject for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12times per year, or a combination thereof.

In some embodiments the oral composition comprising Compound I asdescribed herein is administered before, after, or with a meal. In someembodiments the oral composition described herein is administeredbefore, after, or with a high fat meal. In some embodiments, the oralcomposition described herein is administered before, after, or with astandardized high fat meal. In some embodiments, the high-fat meal is ahigh-calorie, high-fat meal. In some embodiments, the high fat mealfollows the FDA guidance on a high-fat meal. In some embodiments, thehigh-calorie, high-fat meal follows the FDA guidance on a high-fat andhigh-calorie meal. In some embodiments, the high-fat meal comprises afat content of about 50% or greater of total caloric content of themeal. See U.S. Department of Health and Human Services Food and DrugAdministration Center for Drug Evaluation and Research, (2002). Guidancefor Industry: Food-Effect Bioavailability and Fed BioequivalenceStudies. Office of Training and Communications Division of DrugInformation, HFD-240. In some embodiments, the high-calorie, high-fatmeal comprises a fat content of at least 50% of total caloric content ofthe meal and a total of about 800 to about 1000 kilocalorie content.

In some embodiments the oral composition described herein isadministered following an overnight fast. In some embodiments, theovernight fast is at least about 6 hours, at least about 7 hours, atleast about 8 hours, at least about 9 hours, or at least about 10 hours.For example, the oral composition described herein may be administeredfollowing a high-fat meal, high-calorie meal following an overnight fastof at least 10 hours.

Combination Therapy

Compound I, i.e., Compound (P)-I and (M)-I in any molar ratio asdescribed supra and in any form (e.g., free form, crystalline form) asdescribed supra, and oral compositions comprising the same can be usedalone or in combination with other pharmaceutically active compounds, totreat conditions such as those described above. The compound(s) of thepresent invention and other pharmaceutically active compound(s) can beadministered simultaneously (either in the same dosage form or inseparate dosage forms) or sequentially. Accordingly, in one embodiment,the present invention comprises methods for treating a condition byadministering to the subject a therapeutically-effective amount of theoral pharmaceutical composition described herein and one or moreadditional pharmaceutically active compounds.

In certain instances, it may be appropriate to administer thecompositions described here in combination with another pharmaceuticalagent. By way of example only, if one of the side effects experienced bya patient upon receiving the composition herein is hypertension, then itmay be appropriate to administer an anti-hypertensive agent incombination with the initial pharmaceutical agent. Or, by way of exampleonly, the therapeutic effectiveness of the composition described hereinmay be enhanced by administration of an adjuvant (e.g., by itself theadjuvant may only have minimal therapeutic benefit, but in combinationwith another pharmaceutical agent, the overall therapeutic benefit tothe patient is enhanced). Or, by way of example only, the benefitexperienced by a patient may be increased by administering thecomposition described herein with another pharmaceutical agent (whichalso includes a therapeutic regimen) that also has therapeutic benefit.By way of example only, in a treatment for rheumatoid arthritisinvolving administration of the composition described herein, increasedtherapeutic benefit may result by also providing the patient withanother pharmaceutical agent for rheumatoid arthritis. In any case,regardless of the disease, disorder, or condition being treated, theoverall benefit experienced by the patient may simply be additive of thetwo pharmaceutical compositions or the patient may experience asynergistic benefit.

The combination methods, compositions and formulations are not to belimited to the use of only two agents, the use of multiple therapeuticcombinations are also envisioned. It is understood that the dosageregimen to treat, prevent, or ameliorate the condition(s) for whichrelief is sought, is optionally modified in accordance with a variety offactors. These factors include the disorder from which the subjectsuffers, as well as the age, weight, sex, diet, and medical condition ofthe subject. Thus, the dosage regimen actually employed varies widely,in some embodiments, and therefore deviates from the dosage regimens setforth herein.

In any case, the multiple therapeutic compositions (at least one ofwhich is the composition disclosed herein) may be administeredconcomitantly in any order or even simultaneously. If simultaneously,the multiple therapeutic compositions may be provided in a single,unified form, or in multiple forms (by way of example only, either as asingle pill or as two separate pills). One of the therapeuticcompositions may be given in multiple doses, or both may be given asmultiple doses. If not simultaneous, the timing between the multipledoses may be any duration of time ranging from a few minutes to eightweeks or at any interval appropriate to maintain the desired therapeuticefficacy. In some embodiments, the timing between the multiple doses maybe a minute, an hour, six hours, a day, two days, three days, four days,five days, six days, a week, two weeks, three weeks, four weeks, fiveweeks, six weeks, seven weeks or eight weeks.

Thus, in another aspect, certain embodiments provide methods fortreating an inflammatory condition in a human or animal subject in needof such treatment comprising administering to said subject an amount ofan oral composition comprising Compound I as disclosed herein in anamount of 5 mg/day to 300 mg/day to reduce or prevent said condition inthe subject, in combination with at least one additional agent for thetreatment of said disorder that is known in the art. In a relatedaspect, certain embodiments provide the therapeutic compositiondisclosed herein in combination with one or more additionalpharmaceutically active compounds and a pharmaceutically acceptablecarrier for the treatment of the inflammatory condition.

The oral compositions comprising Compound I described herein are alsooptionally used in combination with other therapeutic reagents that areselected for their therapeutic value for the condition to be treated. Ingeneral, the compositions described herein and, in embodiments wherecombinational therapy is employed, other agents do not have to beadministered in the same pharmaceutical composition, and, because ofdifferent physical and chemical characteristics, are optionallyadministered by different routes. The initial administration isgenerally made according to established protocols, and then, based uponthe observed effects, the dosage, modes of administration and times ofadministration subsequently modified.

In some embodiments, the one or more additional pharmaceutically activecompounds is selected from the group consisting of anti-inflammatorydrugs, anti-atherosclerotic drugs, immunosuppressive drugs,immunomodulatory drugs, cytostatic drugs, anti-proliferative agents,angiogenesis inhibitors, kinase inhibitors, cytokine blockers andinhibitors of cell adhesion molecules.

In some embodiments, a subject suffering from or at risk of sufferingfrom an inflammatory condition is administered an oral compositioncomprising Compound I together with one or more agents or compositionsknown for treating an inflammatory condition in any combination.

Specific, non-limiting examples of possible combination therapies forany of the inflammatory conditions listed supra include the oralcomposition comprising Compound I as described herein in combinationwith: (1) corticosteroids, including but not limited to cortisone,dexamethasone, and methylprednisolone; (2) nonsteroidalanti-inflammatory drugs (NSAIDs), including but not limited toibuprofen, naproxen, acetaminophen, aspirin, fenoprofen (NALFON™),flurbiprofen (ANSAID™), ketoprofen, oxaprozin (DAYPRO™), diclofenacsodium (VOLTAREN™), diclofenac potassium (CATAFLAM™), etodolac (LODINE™)indomethacin (INDOCIN™), ketorolac (TORADOL™), sulindac (CLINORIL™),tolmetin (TOLECTIN™), meclofenamate (MECLOMEN™), mefenamic acid(PONSTEL™), nabumetone (RELAFEN™) and piroxicam (FELDENE™); (3)immunosuppressants, including but not limited to methotrexate(RHEUMATREX™), leflunomide (ARAVA™), azathioprine (IMURAN™),cyclosporine (NEORAL™, SANDIMMUNE™), tacrolimus and cyclophosphamide(CYTOXAN™); (4) CD20 blockers, including but not limited to rituximab(RITUXAN™); (5) Tumor Necrosis Factor (TNF) blockers, including but notlimited to etanercept (ENBREL™), infliximab (REMICADE™) and adalimumab(HUMIRA™); (6) interleukin-1 receptor antagonists, including but notlimited to anakinra (KINERET™), rilonocept (ARCALYST™) and canakinumab(ILARIS™); (7) interleukin-6 inhibitors, including but not limited totocilizumab (ACTEMRA™); (8) interleukin-17 inhibitors, including but notlimited to secukinumab (COSENTYX®; AIN457), ixekizumab (TALTZ®) andbrodalimumab; (9) Janus kinase inhibitors, including but not limited totofacitinib; and (10) syk inhibitors, including but not limited tofostamatinib.

Where a subject is suffering from or at risk of suffering fromrheumatoid arthritis or a condition that is associated with rheumatoidarthritis, an oral composition comprising Compound I as described hereinis optionally administered together with one or more agents suitable fortreating rheumatoid arthritis or a condition that is associated withrheumatoid arthritis in any combination. Examples of therapeuticagents/treatments for inflammatory conditions, such as rheumatoidarthritis or a condition that is associated with rheumatoid arthritis,that can be administered in combination with Compound I include, but arenot limited, non-steroidal anti-inflammatory drugs (NSAIDs), steroids(e.g., prednisone), corticosteroids, and disease modifying drugs(DMARDs) such as methotrexate, leflunomide, hydroxychloroquine,sulfasalazine, Janus kinase (JAK) inhibitors (e.g., tofacitinib,upadacitinib, baricitinib, filgotinib, ruxolitinib, oclacitinib,peficitinib, fedratinib, cerdulatinib, gandotinib, lesarturtinib,momelotinib, pacritinib, abrocitinib, and BMS-986165), tumor necrosisfactor inhibitors (e.g., adalimumab, etanercept, golimumab, infliximab,certolizumab), anti-B-cell antibodies (e.g., rituximab), anti-IL-6antibodies (e.g., sarilumab, tocilixumab), interleukin-1 receptor (IL-1)antagonist (e.g., anakinra), and T-cell activation inhibitors (e.g.,abatacept).

In any embodiment, Compound I is administered in combination withmethotrexate for the treatment of rheumatoid arthritis or otherinflammatory condition as described herein.

In any embodiment, Compound I is administered in combination with a JAKinhibitor for the treatment of rheumatoid arthritis or otherinflammatory condition as described herein. Suitable JAK inhibitors foradministering in combination with Compound I for the treatment ofrheumatoid arthritis or any of the other inflammatory conditionsdisclosed herein include, without limitation, tofacitinib, upadacitinib,baricitinib, filgotinib, ruxolitinib, oclacitinib, peficitinib,fedratinib, cerdulatinib, gandotinib, lesarturtinib, momelotinib,pacritinib, abrocitinib, and BMS-986165.

In any embodiment, Compound I is administered in combination with a TYK2inhibitor for the treatment of rheumatoid arthritis or otherinflammatory condition as described herein. Suitable TYK2 inhibitors foradministering in combination with Compound I for the treatment ofrheumatoid arthritis or any of the other inflammatory conditionsdisclosed herein include, without limitation, PF-06826647(1r,3r)-3-(cyanomethyl)-3-(4-(6-(1-methyl-1H-pyrazol-4-yl)pyrazolo[1,5-a]pyrazin-4-yl)-1H-pyrazol-1-yl)cyclobutane-1-carbonitrile),3-pyridazinecarboxamide, tofacitinib, upadacitinib, deucravacitinib(6-(cyclopropanecarbonylamino)-4-[2-methoxy-3-(1-methyl-1,2,4-triazol-3-yl)anilino]-N-(trideuteriomethyl)pyridazine-3-carboxamide),cerdulatinib, AT9283(1-Cyclopropyl-3-(3-(5-(morpholinomethyl)-1H-benzo[d]imidazol-2-yl)-1H-pyrazol-4-yl)urea),Nvp-bsk805.2HCl(4-(2,6-difluoro-4-(3-(1-(piperidin-4-yl)-1H-pyrazol-4-yl)quinoxalin-5-yl)benzyl)morpholinedihydrochloride), S-ruxolitinib, gandotinib, pacritinb, baricitinib,filgotinib, izencitinib, NDI-031301 (Akahane et al., Blood 128:1596,2016), and XL019((2R)-N-[4-[2-(4-morpholin-4-ylanilino)pyrimidin-4-yl]phenyl]pyrrolidine-2-carboxamide),brepocitinib([(1S)-2,2-difluorocyclopropyl]-[(1S,5R)-3-[2-[(1-methylpyrazol-4-yl)amino]pyrimidin-4-yl]-3,8-diazabicyclo[3.2.1]octan-8-yl]methanone),VTX-958 (a selective allosteric TYK2 inhibitor developed by VentyxBiosciences), and NDI-031407 (a selective TYK2 inhibitor developed byNimbus Therapeutics and disclosed in Gracey, E., et al. J. Clin.Invest.; 130(4):1863-1878 (2020, which is hereby incorporated byreference in its entirety).

In any embodiment, Compound I is administered in combination with a BTKinhibitor for the treatment of rheumatoid arthritis or otherinflammatory condition as described herein. Suitable BTK inhibitors foradministering in combination with Compound I for the treatment ofrheumatoid arthritis or any of the other inflammatory conditionsdisclosed herein include, without limitation, ibrutinib, acalabrutinib,fenebrutinib, and zanubrutinib.

In any embodiment, Compound I is administered in combination with anIRAK4, IKKi, tpl2, or CTLA4 inhibitor for the treatment of rheumatoidarthritis or other inflammatory condition as described herein. SuitableIRAK4, IKKi, tpl2, and CTLA4 inhibitors for administering in combinationwith Compound I for the treatment of rheumatoid arthritis or any of theother inflammatory conditions disclosed herein include, withoutlimitation,N-[1-(2-morpholin-4-ylethyl)benzimidazol-2-yl]-3-nitrobenzamide,N-(1-phenyl-5-(piperidin-1-ylmethyl)-1H-benzo[d]imidazol-2-yl)-3-(trifluoromethyl),benzamide, ibrutinib,(R)-6-((1,6-naphthyridin-2-yl)amino)-4-(cyclopropylamino)-N-(2-fluoro-3-hydroxy-3-methylbutyl)nicotinamide,N-(4-morpholin-4-ylcyclohexyl)-5-(oxan-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine,N-(trans-4-morpholinocyclohexyl)-5-(tetrahydro-2H-pyran-4-yl)-7H-pyrrolo[2,3-d]pyrimidin-4-amine,4-[(4-morpholin-4-ylcyclohexyl)amino]quinazoline-6-carbonitrile,1-[[(2S,3S,4S)-3-Ethyl-4-fluoro-5-oxo-2-pyrrolidinyl]methoxy]-7-methoxy-6-isoquinolinecarboxamide,2-(4-fluorophenyl)-6-methyl-4-(3-(trifluoromethyl)phenyl)-1,2-dihydrodipyrazolo[3,4-b:3′,4′-d]pyridin-3(6H)-one,ipilimumab, abatacept,4-(3-chloro-4-fluoroanilino)-6-(pyridin-3-ylmethylamino)-1,7-naphthyridine-3-carbonitrile,6-[7-[3-cyano-4-(oxan-4-yloxy)phenyl]furo[3,2-b]pyridin-2-yl]-5-methoxy-N,N-dimethylpyridine-3-carboxamide.

Specific, non-limiting examples of possible combination therapies forthe treatment of cancer include the oral composition comprising CompoundI as described herein in combination with: (1) alkylating agents,including but not limited to cisplatin (PLATIN™), carboplatin(PARAPLATIN™), oxaliplatin (ELOXATIN™), streptozocin (ZANOSAR™) busulfan(MYLERAN™) and cyclophosphamide (ENDOXAN™); (2) anti-metabolites,including but not limited to mercaptopurine (PURINETHOL™), thioguanine,pentostatin (NIPENT™), cytosine arabinoside (ARA-C™), gemcitabine(GEMZAR™), fluorouracil (CARAC™), leucovorin (FUSILEV™) and methotrexate(RHEUMATREX™); (3) plant alkaloids and terpenoids, including but notlimited to vincristine (ONCOVIN™), vinblastine and paclitaxel (TAXOL™);(4) topoisomerase inhibitors, including but not limited to irinotecan(CAMPTOSAR™), topotecan (HYCAMTIN™) and etoposide (EPOSIN™); (5)cytotoxic antibiotics, including but not limited to actinomycin D(COSMEGEN™), doxorubicin (ADRIAMYCIN™), bleomycin (BLENOXANE™) andmitomycin (MITOSOL™); (6) angiogenesis inhibitors, including but notlimited to sunitinib (SUTENT™) and bevacizumab (AVASTIN™); (7) tyrosinekinase inhibitors, including but not limited to imatinib (GLEEVEC™),erlotinib (TARCEVA™), lapatininb (TYKERB™) and axitinib (INLYTA™); and(8) immune checkpoint inhibitors, including but not limited toatezolizumab (TECENTRIQ™), avelumab (BAVENCIO™), durvalumab (IMFINZI™),ipilimumab (YERVOY™), pembrolizumab (KEYTRUDA™), nivolumab (OPDIVO™),and tremelimumab.

In some embodiments, the oral composition described herein isadministered in combination with an additional therapeutic agentselected from a chemotherapeutic or anti-proliferative agent, antiviral,antibiotic, antihistamine, an emollient, systemic phototherapy, psoralenphotochemotherapy, laser therapy, hormone replacement therapy, ananti-inflammatory agent, an immunomodulatory or immunosuppressive agent,an agent for treating cardiovascular disease, an agent for treatingdiabetes, an agent for treating immunodeficiency disorders, or anycombination thereof.

In any embodiment, Compound I may be administered to a subject havingpsoriatic arthritis in combination with one or more JAK inhibitorsincluding, but limited to, tofacitinib, upadacitinib, baricitinib,filgotinib, ruxolitinib, oclacitinib, peficitinib, fedratinib,cerdulatinib, gandotinib, lesarturtinib, momelotinib, pacritinib,abrocitinib, and BMS-986165, or BTK inhibitors, including, but notlimited to, ibrutinib, acalabrutinib, fenebrutinib, and zanubrutinib. Inany embodiment, Compound I may be administered to a subject havingpsoriatic arthritis in combination with one or more of the following:NSAIDs; one or more DMARDs such as methotrexate, sulfasalazine,leflunomide, abatacept, adalimumab, certolizumab, etanercept, golimumab,infliximab, ixekizumab, secukinumab, tocilizumab, tofacitinib,ustekinumab; one or more immunosuppressants such as azathioprine orcyclosporine; apremilast, or steroid (injection).

In any embodiment, Compound I may be administered to a subject havingpsoriasis in combination with one or more JAK inhibitors, including, butlimited to, tofacitinib, upadacitinib, baricitinib, filgotinib,ruxolitinib, oclacitinib, peficitinib, fedratinib, cerdulatinib,gandotinib, lesarturtinib, momelotinib, pacritinib, abrocitinib, andBMS-986165, or BTK inhibitors including, but not limited to, ibrutinib,acalabrutinib, fenebrutinib, and zanubrutinib. In any embodiment,Compound I may be administered to a subject having psoriasis incombination with one or more of the following: corticosteroids; vitaminD analogues; retinoids; calcineurin inhibitors; salicylic acid;anthralin; cyclosporine; one or more DMARDs such as methotrexate,etanercept, infliximab, adalimumab, ustekinumab, secukinumab,ixekizumab; thioguanine, hydroxyurea, apremilast, coal tar in optionalcombination with light therapy (e.g., Goeckerman therapy), or anynatural or alternative treatments such as aloe extract cream, fish oil,Oregon grape, or essential oils.

In any embodiment, Compound I may be administered to a subject havinghidradenitis suppurativa (HS) in combination with one or more JAKinhibitors, including, but not limited to, tofacitinib, upadacitinib,baricitinib, filgotinib, ruxolitinib, oclacitinib, peficitinib,fedratinib, cerdulatinib, gandotinib, lesarturtinib, momelotinib,pacritinib, abrocitinib, and BMS-986165; tumor necrosis factorinhibitors including, but not limited to, adalimumab, etanercept,golimumab, infliximab, certolizumab; IL-1β and IL-1α inhibitors,including, but not limited to, anakinra, rilonacept, canakinumab,gevokizumab, LY2189102 ((Bihorel et al., AAPS J. 16(5):1009-1117, 2014),and bermekimab. In any embodiment, Compound I may be administered to asubject having hidradenitis suppurativa (HS) in combination with one ormore of the following: antibiotics such as clindamycin, gentamicin,rifampin, doxycycline, minocycline; one or more DMARDs such asmethotrexate, adalimumab, infliximab, anakinra, canakinumab,ustekinumab; one or more NSAIDs; one or more retinoids, such asisotretinoin and acetretin; resorcinol (e.g., topical); one or morehormones such as spirolactone, finasteride; or metformin.

In any embodiment, Compound I may be administered to a subject havingcryopyrin-associated autoinflammatory syndrome (CAPS) in combinationwith one or more interleukin-1 receptor antagonists, including but notlimited to anakinra (KINERET™), rilonocept (ARCALYST™) and canakinumab(ILARIS™). In some embodiments, the combination of Compound I and IL-1antagonist is used to induce remission and then remission is maintainedwith administration of Compound I. In other embodiments, Compound I maybe administered to a subject having cryopyrin-associatedautoinflammatory syndrome (CAPS) in combination with one or more of thefollowing: rilonacept, canakinumab, anakinra, methotrexate, one or moresteroids, or one or more NSAIDs.

These various agents/compositions that are used in combination with theoral compositions described herein can be used in accordance with theirstandard or common dosages, as specified in the prescribing informationaccompanying commercially available forms of the drugs (see also, theprescribing information in the 2006 Edition of The Physician's DeskReference). In some embodiments, standard dosages of these agents may bereduced when used in combination with the oral compositions describedherein. Without limiting the scope of this disclosure, it is believedthe such combination may result in synergistic results with betterefficacy, less toxicity, longer duration of action, or quicker responseto therapy. In some embodiments, the combination therapies inembodiments herein may be administered in sub-therapeutic amounts ofeither the oral compositions described herein or the additionalpharmaceutical agents, or both.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

Embodiments of the Disclosure

The invention provides also the following non-limiting embodiments.

Embodiment is 1 a method for treating an inflammatory condition thatinvolves administering, to a human subject having an inflammatorycondition, an oral dose of 5 mg/day to 300 mg/day of Compound I

or a derivative thereof to treat said inflammatory condition.

Embodiment 2 is the method of Embodiment 1, wherein 50 mg/day of thecompound is administered to said subject.

Embodiment 3 is the method of Embodiment 1, wherein 100 mg/day of thecompound is administered to said subject.

Embodiment 4 is the method of Embodiment 1, wherein 160 mg/day of thecompound is administered to said subject.

Embodiment 5 is the method of Embodiment 1, wherein 200 mg/day of thecompound is administered to said subject.

Embodiment 6 is the method of Embodiment 1, wherein 240 mg/day of thecompound is administered to said subject.

Embodiment 7 is the method of any one of Embodiments 1-6, wherein thecompound is administered once a day.

Embodiment 8 is the method of any one of Embodiment 1-6, wherein thecompound is administered twice a day.

Embodiment 9 is the method of claim 1, wherein 50 mg of the compound isadministered to the subject twice daily.

Embodiment 10 is the method of claim 1, wherein 80 mg of the compound isadministered to the subject twice daily.

Embodiment 11 is the method of Embodiment 1, wherein 120 mg of thecompound is administered to the subject twice daily.

Embodiment 12 is the method of any one of Embodiments 1-11, whereinCompound I is deuterated.

Embodiment 13 is the method of any one of Embodiments claims 1-12,wherein Compound I comprises the Compound (P)-I

and the Compound (M)-I

in a molar ratio of (P)-I to (M)-I of about 4:1.

Embodiment 14 is the method of Embodiment 13, wherein the molar ratio of(P)-I to (M)-I is about 9:1.

Embodiment 15 is the method of Embodiment 13, wherein the molar ratio of(P)-I to (M)-I is about 99:1.

Embodiment 16 is the method of Embodiment 13, wherein the molar ratio of(P)-I to (M)-I is about 199:1.

Embodiment 17 is the method of Embodiment 13, wherein the molar ratio of(P)-I to (M)-I is about 399:1.

Embodiment 18 is the method of Embodiment 13, wherein the molar ratio of(P)-I to (M)-I is about 999:1.

Embodiment 19 is the method of any one of claims 1-12, wherein CompoundI comprises the Compound (P)-I substantially free of Compound (M)-I.

Embodiment 20 is the method of any one of Embodiments claims 1-12,wherein Compound I comprises at least 80 mol % of Compound (P)-I

Embodiment 21 is the method of Embodiment 20, wherein Compound Icomprises at least 90 mol % of Compound (P)-I.

Embodiment 22 is the method of Embodiment 20, wherein Compound Icomprises at least 95 mol % of Compound (P)-I.

Embodiment 23 is the method of Embodiment 20, wherein Compound Icomprises at least 99 mol % of Compound (P)-I.

Embodiment 24 is the method of any one of Embodiments 1-23, whereinCompound (P)-I is a free base.

Embodiment 25 is the method of any one of Embodiments 1-23, whereinCompound (P)-I is a pharmaceutically acceptable salt.

Embodiment 26 is the method of any one of Embodiments 1-23, whereinCompound (P)-I is crystalline form.

Embodiment 27 is the method of Embodiment 26, wherein the crystallineform of Compound (P)-I is crystalline Form A characterized by an PXRDpattern having a peak expressed in degrees 2θ at about 9.78±0.2.

Embodiment 28 is the method of Embodiment 26, wherein the crystallineform of Compound (P)-I is the crystalline Form A characterized by anPXRD pattern having peaks expressed in degrees 2θ at 9.78±0.2 and15.51±0.2.

Embodiment 29 is the method of Embodiment 26, wherein the crystallineform of Compound (P)-I is the crystalline Form A characterized by anPXRD pattern having peaks expressed in degrees 2θ at 9.78±0.2,15.51±0.2, 19.6±0.2, and 25.92±0.2.

Embodiment 30 is the method of Embodiment 26, wherein the crystallineform of Compound (P)-I is the crystalline Form A characterized by anPXRD pattern having peaks expressed in degrees 2θ at 9.78±0.2,15.34±0.2, 15.51±0.2, 19.6±0.2, 20.57±0.2, 21.01±0.2, 25.92±0.2,29.05±0.2, and 29.48±0.2,

Embodiment 31 is the method of any one of Embodiments 1-30, wherein theinflammatory condition is rheumatoid arthritis.

Embodiment 32 is the method of any one of Embodiments 1-30, wherein theinflammatory condition is psoriatic arthritis.

Embodiment 33 is the method of any one of Embodiments 1-30, wherein theinflammatory condition is a cryopyrin-associate periodic syndrome.

Embodiment 34 is the method of any one of Embodiments 1-30, wherein theinflammatory condition is Hidradenitis suppurativa.

Embodiment 35 is the method of any one of Embodiments 1-30, wherein theinflammatory condition is selected from the group consisting ofpsoriasis, juvenile idiopathic arthritis, ulcerative colitis, Crohn'sdisease, ankylosing spondylitis, pancreatic cancer, metastatic breastcancer, gout, recurrent pericarditis, and idiopathic pulmonary fibrosis.

Embodiment 36 is the method of any one of Embodiments 1-30, wherein theinflammatory condition is selected from spondyloarthritis such asankylosing spondylitis, psoriatic arthritis, reactive arthritis andReiter's syndrome, juvenile rheumatoid arthritis (JIA), systemic-onsetjuvenile rheumatoid arthritis, idiopathic arthritis (JIA) (includingsystemic (SJIA)), and gout; cryopyrin-associated autoinflammatorysyndromes (CAPS), including Muckle-Wells syndrome (MWS), neonatal-onsetmultisystem inflammatory disease (NOMID), and familial coldautoinflammatory syndrome (FCAS); chronic obstructive pulmonary diseases(COPD), including emphysema, chronic bronchitis, and asthma (allergicand non-allergic), hidradenitis suppurativa (HS); psoriasis, such asplaque psoriasis; colitis from an inflammatory bowel disease (IBD) likeCrohn's disease or ulcerative colitis and inflammatory boweldisease-associated arthritis; pericarditis, including acutepericarditis, recurrent pericarditis, and chronic pericarditis;pulmonary inflammation or fibrosis, including idiopathic pulmonaryfibrosis; metastatic breast cancer, and pancreatic cancer.

Embodiment 37 is the method of any one of Embodiments 1-30, wherein theinflammatory condition is selected from Familial Mediterranean Fever(FMF); tumor necrosis factor receptor-associated periodic syndrome(TRAPS); adult-onset Still's disease; pyoderma gangrenosum;bone-resorption disorders (such as those associated with cancer (e.g.,breast cancer)); metastatic melanoma; Castleman disease; and chronicatypical neutrophilic dermatosis with lipodystrophy (CANDLE).

Embodiment 38 is the method of any one of Embodiments 1-30, wherein theinflammatory condition is pruritus, which may be associated with anyother condition, for example, pruritus associated with hidradenitissuppurativa, pruritus associated with inflammation, pruritus associatedwith rheumatoid arthritis, pruritus associated with psoriasis, andpruritus associated with TH17-associated inflammation.

Embodiment 39 is the method of any one of Embodiments 1-30, wherein theinflammatory condition is selected from Lyme disease; cytokine releasesyndrome (CRS); acute respiratory distress syndrome (ARDS); chronic oracute bronchitis; epidermolysis bullosa (EB); bullous pemphigoid;juvenile dermatomyositis; inflammatory vitiligo (including marginal);pemphigus vulgaris; enterocolitis; polymyositis; myositis, bone cancer;lung cancer; inflammatory bone disorders such as chronic recurrent multiosteomyelitis (CRMO), Synovitis, acne, pustulosis, hyperostosis, andosteitis (SAPHO) syndrome, Majeed syndrome, deficiency of interleukin-1receptor antagonist (DIRA) and cherubism; bone resorption (such as isassociated with an autoimmune disease); neuroinflammatory diseases suchas Alzheimer's disease (AD), Parkinson's disease (PD), multiplesclerosis (MS), acute disseminated encephalomyelitis (ADEM), acute opticneuritis (AON), transverse myelitis, and neuromyelitis optical (NMO);Behcet's disease; endotoxic shock (e.g., toxic shock syndrome (TSS) andother systemic gram-negative bacterial infections); enthesitis;polyarteritis nodosa (PAN); chronic pain; polymyalgia rheumatica;chronic allograft rejection; Sjogren's syndrome; and Schnitzler'ssyndrome (SchS).

Embodiment 40 is the method of any one of Embodiments 1-30, wherein 50mg of the compound is administered to a subject having rheumatoidarthritis twice daily.

Embodiment 41 is the method of any one of Embodiments 1-30, wherein 80mg of the compound is administered to a subject having rheumatoidarthritis twice daily.

Embodiment 42 is the method of any one of Embodiments 1-30, wherein 120mg of the compound is administered to a subject having rheumatoidarthritis twice daily.

Embodiment 43 is the method of any one of Embodiments 36-38, whereinsaid administering is carried out under conditions effective to inhibitprogression of joint damage, improve synovitis, or both in said subjectas assessed by magnetic resonance imaging (MRI).

Embodiment 44 is the method of any one of Embodiments 1-43, wherein saidadministering is carried out under conditions effective to significantlyreduce in vivo serum levels of one or more inflammatory cytokines ascompared to in vivo serum levels of the one or more inflammatorycytokines in a subject administered a placebo.

Embodiment 45 is the method of Embodiment 44, wherein the one or moreinflammatory cytokines is selected from the group consisting of TNF-α,IL-1β, IL-6, IL-8, IFNγ, IL-17, IL-18, IL-1α and MIP1β.

Embodiment 46 is the method of any one of Embodiments 1-45, wherein saidadministering is carried out for greater than 12 weeks withouttachyphylaxis.

Embodiment 47 is the method of any one of Embodiment 1-45, whereinadministering further comprises: administering one or more additionaltherapeutic agents in conjunction with Compound I.

Embodiment 48 is the method of Embodiment 47, wherein said one or moreadditional therapeutic agents is administered simultaneously withCompound I.

Embodiment 49 is the method of Embodiment 47, wherein said one or moreadditional therapeutic agents is administered sequentially with CompoundI.

Embodiment 50 is the method of any one of Embodiments 47-49, wherein theone or more additional therapeutic agents is selected from the groupconsisting of an anti-inflammatory drug, an anti-atherosclerotic drug,an immunosuppressive drug, an immunomodulatory drug, a cytostatic drug,an angiogenesis inhibitor, a kinase inhibitor, a cytokine blocker, andan inhibitor of cell adhesion molecules.

Embodiment 51 is the method of any one of Embodiments 47-49, whereinCompound I as defined in any one of Embodiments 2-30 is administered toa subject having rheumatoid arthritis in conjunction with a JAKinhibitor.

Embodiment 52 is the method of any one of Embodiments 47-49, whereinCompound I as defined in any one of Embodiments 2-30 is administered toa subject having rheumatoid arthritis in conjunction with a TYK2inhibitor.

Embodiment 53 is the method of any one of Embodiments 47-49, whereinCompound I as defined in any one of Embodiments 2-30 is administered toa subject having rheumatoid arthritis in conjunction with a BTKinhibitor.

Embodiment 54 is the method of any one of Embodiments 47-49, whereinCompound I as defined in any one of Embodiments 2-30 is administered toa subject having rheumatoid arthritis in conjunction with a IRAK4inhibitor.

Embodiment 55 is the method of any one of Embodiments 47-49, whereinCompound I as defined in any one of Embodiments 2-30 is administered toa subject having rheumatoid arthritis in conjunction with a IKKiinhibitor.

Embodiment 56 is the method of any one of Embodiments 47-49, whereinCompound I as defined in any one of Embodiments 2-30 is administered toa subject having rheumatoid arthritis in conjunction with a tpl2inhibitor.

Embodiment 57 is the method of any one of Embodiments 47-49, whereinCompound I as defined in any one of Embodiments 2-30 is administered toa subject having rheumatoid arthritis in conjunction with a CTLA4inhibitor.

Embodiment 58 is the method of any one of Embodiments 1-57, wherein thecompound is formulated as a solid dosage form selected from a tablet, acapsule, a lozenge, a sachet, a powder, granules, and orally dispersiblefilm.

Embodiment 59 is the method of Embodiment 58, wherein the solid dosageform is a tablet.

Embodiment 60 is the method of any one of Embodiments 1-59, wherein thecompound is administered as an immediate release formulation.

Embodiment 61 is the method of any one of Embodiment 1-59, wherein thecompound is administered as a controlled release formulation.

Embodiment 62 is an oral pharmaceutical composition comprising: CompoundI

or a derivative thereof in an amount of 5 mg to 300 mg, and apharmaceutically acceptable carrier.

Embodiment 63 is the oral composition of Embodiment 62, wherein saidcomposition comprises 50 mg of Compound I.

Embodiment 64 is the oral composition of Embodiment 62, wherein saidcomposition comprises 80 mg of Compound I.

Embodiment 65 is the oral composition of Embodiment 62, wherein saidcomposition comprises 100 mg of Compound I.

Embodiment 66 is the oral composition of Embodiment 62, wherein saidcomposition comprises 120 mg of Compound I.

Embodiment 67 is the oral composition of Embodiment 62, wherein saidcomposition comprises 160 mg of Compound I.

Embodiment 68 is the oral composition of Embodiment 62, wherein saidcomposition comprises 200 mg of Compound I.

Embodiment 69 is the oral composition of Embodiment 62, wherein saidcomposition comprises 240 mg of Compound I.

Embodiment 70 is the oral composition of any one of Embodiments 62-69,wherein Compound I is deuterated.

Embodiment 71 is the oral composition of any one of Embodiments 62-69wherein Compound I comprises the Compound (P)-I

and the Compound (M)-I

in a molar ratio of (P)-I to (M)-I of about 4:1.

Embodiment 72 is the oral composition of Embodiment 71, wherein themolar ratio of (P)-I to (M)-I is about 9:1.

Embodiment 73 is the oral composition of Embodiment 71, wherein themolar ratio of (P)-I to (M)-I is about 99:1.

Embodiment 74 is the oral composition of Embodiment 71, wherein themolar ratio of (P)-I to (M)-I is about 199:1.

Embodiment 75 is the oral composition of Embodiment 71, wherein themolar ratio of (P)-I to (M)-I is about 399:1.

Embodiment 76 is the oral composition of any one of Embodiments 62-69,wherein Compound I comprises the Compound (P)-I substantially free ofCompound (M)-I.

Embodiment 77 is the oral composition of any one of Embodiments 62-69,wherein Compound I comprises at least 80 mol % of Compound (P)-I

Embodiment 78 is the oral composition of Embodiments 62-69, whereinCompound I comprises at least 90 mol % of Compound (P)-I.

Embodiment 79 is the oral composition of Embodiments 62-69, whereinCompound I comprises at least 95 mol % of Compound (P)-I.

Embodiment 80 is the oral composition of Embodiments 62-69, whereinCompound I comprises at least 99 mol % of Compound (P)-I.

Embodiment 81 is the oral composition of any one of Embodiments 62-80,wherein Compound I (P)-I is a free base.

Embodiment 82 is the oral composition of any one of Embodiments 62-80,wherein Compound I (P)-I is a pharmaceutically acceptable salt.

Embodiment 83 is the oral composition of any one of Embodiments 62-80,wherein Compound I (P)-I is crystalline form.

Embodiment 84 is the oral composition of Embodiment 83, wherein thecrystalline form of Compound (P)-I is crystalline Form A characterizedby an PXRD pattern having a peak expressed in degrees 2θ at about9.78±0.2.

Embodiment 85 is the oral composition of Embodiment 83, wherein thecrystalline form of Compound (P)-I is the crystalline Form Acharacterized by an PXRD pattern having peaks expressed in degrees 2θ at9.78±0.2 and 15.51±0.2.

Embodiment 86 is the oral composition of Embodiment 83, wherein thecrystalline form of Compound (P)-I is the crystalline Form Acharacterized by an PXRD pattern having peaks expressed in degrees 2θ at9.78±0.2, 15.51±0.2, 19.6±0.2, and 25.92±0.2.

Embodiment 87 is the oral composition of Embodiment 83, wherein thecrystalline form of Compound (P)-I is the crystalline Form Acharacterized by an PXRD pattern having peaks expressed in degrees 2θ at9.78±0.2, 15.34±0.2, 15.51±0.2, 19.6±0.2, 20.57±0.2, 21.01±0.2,25.92±0.2, 29.05±0.2, and 29.48±0.2,

Embodiment 88 is the oral composition of any one of Embodiments 62-87,wherein said composition is formulated into a solid dosage form selectedfrom a tablet, a capsule, a lozenge, a sachet, a powder, granules, andan orally dispersible film.

Embodiment 89 is the oral composition of any one of Embodiments 62-87,wherein said composition is formulated into a tablet.

Embodiment 90 is the oral composition of any one of Embodiments 62-87,wherein the composition is an immediate release formulation.

Embodiment 91 is the oral composition of any one of Embodiments 62-87,wherein the composition is a controlled release formulation.

EXAMPLES

As described in detail above,3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-2′-(2-(2-hydroxypropan-2-yl)pyrimidin-4-yl)-5′,6-dimethyl-2H-[1,4′-bipyridin]-2-one(i.e., Compound I) exists as a P atropisomer and an M atropisomer. Inthe Examples and Figures of this disclosure, “ATI-450” refers to theaforementioned compound containing about 99.75 mol % of the Patropisomer and 0.25 mol % of the M atropisomer.

Example 1 Safety, Pharmacokinetics and Pharmacodynamics of OrallyAdministered ATI-450

ATI-450 is an oral, small molecule inhibitor of the p38αmitogen-activated protein kinase (MAPK)/MAPK-activated protein kinase 2(MK2) inflammatory signaling pathway. This Phase 1, single and multipleascending dose (SAD, MAD), study evaluated ATI-450for safety,tolerability, pharmacokinetics, and pharmacodynamics. Healthy adultswere randomly assigned to SAD (10, 30, 50, 100 mg; n=24) and MAD (10,30, 50 mg twice daily (BID) for 7 days; n=24) cohorts of ATI-450 orplacebo (n=14). Safety and tolerability were evaluated through clinicaland laboratory assessments. Blood PK parameters were determined andpharmacodynamic inhibition of ex vivo, endotoxin-induced tumor necrosisfactor a (TNF-α), interleukin (IL)-1β, IL-6, IL-8, and phosphorylationof the MK2 substrate, phosphorylated heat shock protein 27 (p-HSP27) wasquantitated. The most common adverse events were headache (10/48,20.8%), dizziness (6/48, 12.5%), upper respiratory tract infection(3/48, 6.3%), and constipation (3/48, 6.3%). ATI-450 had doseproportional pharmacokinetics with a terminal half-life of 9-12 hours inthe MAD cohorts on day 7. Dose- and concentration-dependent inhibitionof ex vivo stimulated cytokines and target biomarker was observed. Onday 7, patients in the 50 mg BID dose cohort recorded mean trough druglevels that were 1.4, 2.5, 2.5, and 2.4 times greater than the IC₅₀ forTNF-α, IL-1β, IL-8, and p-HSP27, respectively. Mean C_(max) drug levelswere 3.6, 6.4, 6.2, and 6.0 times greater than the IC₈₀ for TNF-α,IL-1β, IL-8, and p-HSP27, respectively. IL-6 inhibition >50% was notedfor part of the dosing interval. These results support further study ofATI-450 in immuno-inflammatory diseases.

The p38 mitogen-activated protein kinase (p38MAPK) signaling pathway hasbeen a target for therapeutic intervention for inflammatory diseasesbecause of its involvement in the regulation and expression of multiplecytokines (e.g., tumor necrosis factor α [TNF-α], interleukin [IL] 1β,and IL-6) and other inflammatory signals. Small-molecule inhibitors ofp38MAPK have been evaluated in clinical trials for the treatment ofinflammatory diseases; however, clinical development of these compoundshas been limited by toxicity and lack of sustained efficacy. A keychallenge with global p38MAPK inhibition is its ubiquitous expressionand the broad effects it exerts on cellular physiology as a consequenceof the more than 60 substrates that it regulates. Throughphosphorylation of these substrates, p38MAPK regulates negative feedbackloops and anti-inflammatory pathways, which are both involved indownregulating inflammation; thus, global blockade of p38MAPK pathwaysmay inhibit both pro-inflammatory and anti-inflammatory pathways,limiting its potential efficacy in some disease states. Furthermore,several p38MAPK substrates are involved in the regulation of cellularfunction and inhibition of these proteins may result in toxicity.

The clinical efficacy achieved with p38MAPK inhibitors has beengenerally disappointing. Of note, there is evidence in certainautoimmune diseases that p38MAPK inhibitors exhibit tachyphylaxis aftermultiple treatments, making sustained inhibition of the inflammatoryresponse difficult to achieve in these therapeutic indications.Tachyphylaxis may be explained by the negative feedback and/oranti-inflammatory target substrates described above. Therefore, theapproach of targeting key downstream kinase substrates of p38MAPK thatspecifically regulate inflammatory cytokines to potentially achievegreater specificity for blockade of pathological inflammation has gainedinterest. MAPK-activated protein kinase 2 (MK2) is a direct downstreamsubstrate of p38MAPK, which has been recognized as a key driver ofinflammation. Inflammatory cytokines activated through the p38MAPK/MK2pathway include TNF-α, IL-1β, IL-6, and IL-8.

The novel approach of selectively inhibiting the p38MAPK/MK2biomolecular complex instead of p38MAPK alone results in effectiveblockade of the proinflammatory axis while sparing the anti-inflammatorypathways, negative feedback substrates, and proteins that regulategeneral cellular function. This approach has the potential to generatesafer compounds with greater and more sustained anti-inflammatoryefficacy compared with global p38MAPK inhibitors. Unfortunately,directly targeting MK2 has been largely unsuccessful.

ATI-450, a recently developed MK2 inhibitor, has a novel mechanism ofaction by which it targets the modified p38MAPK ATP binding pocket andjuxtaposed MK2 formed upon the interaction between p38MAPK and MK2. Thep38MAPK binding site on MK2 is located within the C terminus;thermodynamic studies have shown that the 2 proteins in the complex bindtightly, and deletion of the binding sequence in MK2 abrogatesp38-dependent phosphorylation and activation of MK2. Following theformation of the p38MAPK-MK2 biomolecular complex, ATI-450 binds to theinterface of the complex with significantly higher affinity than toeither kinase alone, thereby selectively inhibiting p38MAPKphosphorylation of MK2 and locking MK2 in an inactive conformation. MK2regulates inflammatory cytokine mRNA stability and translation throughphosphorylation of downstream effectors includingadenylate-uridylate-rich element-binding proteins (e.g.,tristetraprolin). Therefore, inhibition of p38αMAPK-MK2 blocksdownstream MK2-mediated inflammatory actions.

Preclinical animal disease models have been used to predict thepotential of ATI-450 as a treatment in several immune-mediatedinflammatory diseases. In a rat streptococcal cell wall arthritis modelfor rheumatoid arthritis, ATI-450 showed joint protective effects andpreserved bone mineral density. ATI-450 also was studied in a murinemodel of neonatal-onset multisystem inflammatory disease (NOMID), whichis a severe form of a cryopyrin-associated periodic syndrome; NOMID andother cryopyrin-associated periodic syndromes comprise a spectrum ofrare hereditary autoinflammatory disorders in which IL-1β and IL-18 areoverproduced. A dramatic attenuation of disease was observed in thistransgenic animal model, including a reduction in bone marrow levels ofIL-1β in mice treated with ATI-450 relative to those treated withplacebo. These preclinical results support clinical development ofATI-450 in humans for autoimmune and autoinflammatory diseases driven bythe cytokines TNF-α, IL-1β and IL-6.

As described herein, a first-in-human, Phase 1, randomized,observer-blind, placebo-controlled study was conducted to evaluate thesafety, tolerability, PK, and pharmacodynamic (PD) of ATI-450 in singleand multiple ascending dose (SAD and MAD) cohorts in healthy subjects.In addition, the study included separate cohorts that evaluated the PKof fed versus fasting administration and of co-administration withmethotrexate. Herein is reported the data from the SAD and MAD cohorts.

Results

Study Subjects. In the SAD cohorts, 32 subjects were enrolled,randomized, and included in the safety set. A total of 31 subjectscompleted the study; 1 subject in the 30 mg ATI-450 cohort withdrew fromthe study for non-safety reasons after receiving a single dose on day 1and thus did not complete all study assessments. PK and PD analysesincluded the 24 subjects who received ATI-450. The majority of subjectsin the SAD cohorts were female (84%; Table 1) and ages ranged from 18 to51 years.

In the MAD cohorts, 30 subjects were enrolled, randomized, and completedthe study, and all subjects were included in the safety set; the 24subjects who received ATI-450 were included in the PK and PD sets. Thesecohorts were more evenly split between female and male subjects (57%female, 43% male) and ages ranged from 21 to 53 years.

Safety and Tolerability. No clinically significant findings in vitalsigns, electrocardiograms, or clinical laboratory values were observed.The most common treatment-emergent adverse events (TEAEs) among allstudy participants are shown in Table 2. In the SAD cohorts, 15 TEAEswere reported by 7 of 24 (29%) subjects who received ATI-450 and 6 TEAEswere reported by 4 of 8 (50%) subjects who received placebo. No deathsor serious adverse events (SAEs) and no discontinuations due to TEAEsoccurred. All TEAEs were mild, transient, and resolved without sequelaeby the end of the study. Four of 24 (17%) subjects who received ATI 450reported TEAEs that were considered by the investigator to be related tothe study drug (0 subjects in the 10 mg cohort; 2 [33%] subjects in the30 mg cohort [1 event of nausea and 2 events each of headache anddizziness]; 1 [17%] subject in the 50 mg cohort [1 event of headache];and 1 subject in the 100 mg cohort [2 events of dizziness]).

In the MAD cohorts, there were 24 TEAEs reported by 12 of 24 (50%)subjects who received ATI-450 and 3 TEAEs reported by 3 of 6 (50%)subjects who received placebo. As in the SAD cohorts, there were nodeaths or SAEs and no subjects discontinued the study due to TEAEs; allTEAEs were mild, transient, and resolved without sequelae by the nextvisit. Nine of 24 (38%) subjects who received ATI-450 reported TEAEsthat were considered by the investigator to be related to the study drug(1 of 8 [13%] subjects in the 10 mg BID cohort [1 event of headache], 5of 8 [63%] subjects in the 30 mg BID cohort [1 event each of blurredvision, constipation, abdominal pain, diarrhea, arthralgia, dizziness,and oropharyngeal pain], and 3 of 8 [38%] subjects in the 50 mg BIDcohort [1 event of headache and 2 events of dizziness]). In the placebogroup, 2 of 6 (33%) subjects had TEAEs that were considered by theinvestigator to be related to the study drug (1 event each of vomitingand headache).

Laboratory changes were generally unremarkable. There was a dosedependent reduction in neutrophils observed with the maximum effect inthe MAD cohort observed at Day 2 to Day 5. The mean reduction at day 7in the 50 mg BID group was 24% (3318.4 cells/μL at baseline versus2514.4 cells/μL at day 7).

Pharmacokinetics—SAD cohorts. As shown in FIG. 1, administration ofATI-450 at single doses of 10 mg, 30 mg, 50 mg, and 100 mg under fastingconditions resulted in ATI-450 post-dose mean plasma concentrationsabove the lower limit of quantification through 24 hours after dosing at10 mg and through 48 hours after dosing at all other dose levels.ATI-450 mean plasma concentrations increased with increasing doses (FIG.1). Systemic exposure increased in a dose-proportional manner across theATI-450 dose range from 10 to 100 mg, with approximately 10.8-, 12.6-,and 12.8-fold increases in Cmax, AUC0-t, and AUC0-inf, respectively(Table 3). Statistical analysis for dose proportionality using the powermodel is shown in Table 8, where systemic exposure (C_(max), AUC_(0−t),and AUC0−∞) increased in proportion to ATI-450 dose after single dosingas indicated by the slope estimates near unity and the 90% CIs includingunity. ATI-450 concentrations appeared rapidly in plasma, with medianT_(max) ranging from 2 to 4 hours across the dose cohorts. Mean t_(1/2)of ATI-450 in plasma ranged from 8.5 to 11.2 hours. C_(L/F), T_(max),and t_(1/2) values showed no clear relation with dose over the doserange of 10 mg to 100 mg. Across the dose cohorts, low inter-subjectvariability in C_(max), AUC_(0−∞), and AUC_(0−t) was observed withcoefficients of variation ranging from 12.7% to 28.1%.

Table 1, shown below, summarizes the pharmacokinetic data for the SADcohort fed versus fasting subjects.

TABLE 1 Mean (SD) Pharmacokinetic Parameters of ATI-450 after SingleOral Doses to Healthy Subjects in the Fasted or Fed State (n = 6 perDose Cohort) Dose Fed/Fasted C_(max) T_(max) ^(a) AUC_(0-∞) t_(1/2) (mg)State (ng/mL) (h) (ng · h/mL) (h) 10 Fasted 39.4 (10.4) 2.0 (2.0, 4.0)285.3 (78.4) 8.5 (3.2) 30 Fasted 122.0 (33.4) 4.0 (2.0, 4.1) 1102.4(247.4) 10.7 (3.3)  50 Fasted 160.7 (20.4) 3.0 (2.0, 4.0) 1464.7 (269.5)9.1 (2.4) 100 Fasted 426.0 (110.6) 2.0 (2.0, 4.0) 3654.8 (522.9) 11.2(5.8)  100 Fed^(b) 370.7 (101.0)  6.0 (4.0, 12.0)  4098.5 (1177.2) 6.3(1.6) ^(a)Values are median (min, max) ^(b)Subjects received astandardized high-fat, high-calorie breakfast 30 minutes before doseadministration.

In the fasted state, ATI-450 was rapidly absorbed (T_(max) of 2.0 hoursto 4.0 hours). Approximately dose-proportional increases in mean C_(max)and AUC were observed, indicating linear PK. Moderately slow elimination(terminal t_(1/2) of about 9 to 11 hours) was observed. In the fedstate, absorption was delayed (median T_(max) of 6.0 hours versus 2.0hours), but there did not appear to be any appreciable impact onsystemic exposure to ATI-450 (C_(max) was about 9% lower and AUC_(0−∞)was about 24% higher in the fed state).

Pharmacokinetics—MAD cohorts. FIG. 2 shows the mean (SD) plasmaconcentration-time profiles of ATI-450 from 0 hours to 72 hours afterthe last dose on day 7, representing dose-proportional PK for the MADcohorts, and Table 3 shows ATI-450 PK parameters on day 7 after multipledoses. The results obtained after the first dose on day 1 in the MADcohorts were consistent with those obtained after single doses in theSAD cohorts (data not shown). Tmax appeared to be independent of dose,with median values ranging from 2 to 3 hours following the final doseadministration on day 7. Mean t_(1/2) was similar across doses followingrepeated dose administration (from 9.6 to 11.6 hours). AUC_(tau) andC_(max) increased with escalating doses and there was slightaccumulation after multiple doses. Over the dose range of 10 mg to 50 mgBID, C_(max), and AUC_(tau) increased 5.4- and 5.9-fold on day 1,respectively, and 4.2- and 5.2-fold on day 7, respectively. Steady stateappeared to be reached by day 2 of administration at all dose levelsbased on the similarity in the mean C_(trough) of ATI-450 on days 2through 6. Mean accumulation of ATI-450 after 7 days of BID dosingranged from 1.17- to 1.57-fold for C_(max) and 1.29- to 1.44-fold forAUC_(tau). The mean values of t_(1/2), CL, and apparent volume ofdistribution after multiple doses were comparable to those after asingle dose, indicating time-independent PK of ATI-450 after 7 days ofBID administration. Statistical analysis of dose proportionality on day7 using the power model is shown in Table 9, where systemic exposure(C_(max), AUC_(tau)) following BID administration of ATI-450 increasedin proportion to dose over the 5-fold dose range as indicated by theslope estimates near unity and the 90% CIs including unity.

Table 2 below summarizes the PK parameters of ATI-450 on days 1 and 7 ofdosing at 10, 30, or 50 mg BID.

TABLE 2 Mean (SD) Pharmacokinetic Parameters of ATI-450 afterTwice-daily Oral Doses to Healthy Subjects in the Fasted State (n = 8per Dose Cohort) Dose C_(max) T_(max) ^(a) AUC₀₋₁₂ t_(1/2) Day (mg)(ng/mL) (h) (ng · h/mL) (h) 1 10 34.3 (5.7) 2.0 (2.0, 4.0) 199.5 (35.9) NC^(b) 30 126.5 (21.4) 2.0 (1.0, 4.0) 680.8 (98.8) NC 50 186.7 (66.8)2.0 (2.0, 4.0) 1179.5 (340.1) NC 7 10 51.8 (15.8) 2.0 (2.0, 2.0) 287.8(81.5)  9.6 (2.1) 30 146.5 (33.6) 2.0 (1.0, 4.0) 908.9 (163.1) 10.3(3.2) 50 219.0 (77.8) 3.0 (1.0, 4.0) 1507.8 (659.9) 11.6 (3.7)^(a)Values are median (min, max) ^(b)NC: Not calculable

The PK on day 1 in the MAD cohorts was consistent with PK from the SADcohorts. Linear (dose-and time-independent) PK after multiple-dosing wasobserved. Moderately slow elimination (terminal t_(1/2) of about 9-12hours) was also observed. The trough concentrations were generallysimilar on days 2 through 7, suggesting that the subjects were at ornear steady-state by day 2 of twice-daily administration. Small amountof accumulation (up to 1.4-fold) after multiple dosing

Table 3 below summarizes the PK parameters in subjects dosed withmethotrexate alone or in combination with ATI-450 as shown.

TABLE 3 Mean (SD) Pharmacokinetic Parameters of ATI-450 and Methotrexateafter Oral Administration Alone or in Combination to Healthy MaleSubjects in the Fasted State (n = 15) Dose C_(max) T_(max) ^(b) AUC₀₋₁₂AUC₀₋ _(∞) t_(1/2) Drug (mg) Treatment^(a) (ng/mL) (h) (ng · h/mL) (ng ·h/mL) (h) MTX 7.5 MTX 163.3 (35.6) 1.0 (0.5, 2.5) NA 562.2 (102.2) 3.1(0.7) 7.5 MTX + 142.6 (18.6) 1.5 (1.0, 2.5) NA 518.9 (70.2)  3.0 (0.6)ATI-450 ATI-450 50 ATI-450 ^(c) 219.0 (77.8) 3.0 (1.0, 4.0) 1507.8(659.9) NA NC BID 50 ATI-450 + 182.1 (31.5) 2.0 (1.0, 4.0) 1089.5(262.2) NA NC BID MTX ^(a)Subjects received single 7.5-mg oral doses ofMTX on days 1 and 8, single 15-mg oral doses of leucovorin on days 2 and9, and twice-daily 50-mg oral doses of ATI-450 on days 3-9. ^(b)Valuesare median (min, max). ^(c) Values are from the group of subjects (n =8) administered twice-daily oral doses of ATI-450 at 50 mg during theMAD portion of the study. MTX: Methotrexate; NA: Not applicable; NC: Notcalculable.

The pharmacokinetics of MTX was similar with or without ATI-450exposure. Systemic exposure to ATI-450 appeared to be slightly decreased(about 17% for C_(max) and about 27% for AUC) in the presence of MTXbased on a between-group comparison to the 50-mg ATI-450 data from theMAD portion of the study.

Pharmacodynamics

A relationship between concentrations of each analyte and ATI-450 plasmalevels was developed and the data were then fit to a nonlinearinhibitory E_(max) model, from which IC₅₀ and IC₈₀ concentrations wereestablished. The p-HSP27 and cytokine inhibition parameters generatedfrom the model are shown in Table 8. Target modulation for ATI-450 wassimilar to potencies for inhibition of production of 3 of the 4cytokines, TNF-α, IL-1β, and IL-8, but the potency for inhibition ofIL-6 production was lower (less potent). Imax was greatest for p-HSP27,TNF-α, and IL-6, with greater than 96% inhibition, and lower for IL-1β(74%) and IL-8 (57%).

For the 50 mg BID dose MAD cohort, concentrations at C_(trough) were inexcess of the relative IC₈₀ concentrations (1.4- to 2.4-fold) for thetarget biomarker p-HSP27 and for 3 of the 4 cytokines (TNF-α, IL-1β, andIL-8), but not for IL-6 (Table 4). TNF-α and IL-1β were inhibited by upto 92.7% and 83.0%, respectively, versus baseline. Plasma levels weregreater than the IC₅₀ for IL-6 for at least part of the dosing interval.The effects of ATI-450 dose on the relative concentration of eachcytokine analyte and the biomarker p-HSP27 in the ex vivo stimulatedassay, expressed as a percentage of pre-dosing analyte levels (set to100%) for the 10 mg, 30 mg, and 50 mg BID MAD cohorts on day 7, 4 hoursafter dosing and 12 hours after dosing, are shown in FIG. 4A-4E,respectively. The 4-hour after dosing day 7 samples reflectedapproximate steady-state C_(max) ATI-450 concentrations while the12-hour after dosing day 7 samples reflected steady-state C_(trough)concentrations of the drug. A marked dose-dependent reduction inconcentration was observed for all 4 cytokines at the 4-hour time pointand persisted through 12 hours. TNF-α, IL-1β, IL-8, and p-HSP27 alldemonstrated a reduction in concentration that persisted for the entiredosing interval. Concentration-dependent and dose-dependent modulationof p-HSP27 and inhibition of the 4 cytokines analyzed were demonstrated.

The graph of FIG. 3 shows the inhibition of LPS-stimulated cytokine andchemokine production in Day 7 blood samples taken from subjects dosedwith 50 mg BID of ATI-450 (MAD cohort).

Discussion

Administration of ATI-450 was generally safe and well tolerated byhealthy subjects over a range of single doses up to 100 mg and multipledoses up to 50 mg BID for 7 days. All TEAEs resolved without sequelaeand there were no discontinuations due to adverse events. Dizzinessoccurred more frequently in the active arms but there was no clear doseresponse; the events were generally transient in nature and almost allresolved while on study drug. A dose dependent reduction in neutrophilswas observed and was consistent with the mechanism of action of ATI-450.PK parameters demonstrated that plasma concentrations of ATI-450increased in a dose-proportional manner after administration of singleor multiple ascending doses, which was also confirmed by statisticalanalysis with the power model for both SAD and MAD cohorts. Across allSAD and MAD cohorts, median Tmax ranged from 2 to 4 hours and meant_(1/2) ranged from approximately 9 to 12 hours with no clearrelationship to dose. ATI-450 demonstrated concentration-dependent anddose-dependent modulation of the target biomarker p-HSP27 and inhibitionof the production of TNF-α, IL-1β, IL-6, and IL-8.

ATI-450 presents a novel mechanism by which to inhibit MK2 and thepro-inflammatory cytokines activated by the p38MAPK pathway. As anorally administered drug, ATI-450 provides a potential alternative toinjectable biologic medications that treat immune-mediated inflammatorydiseases. ATI-450 has demonstrated efficacy in animal models for variousimmune-mediated inflammatory diseases and some types of cancer.^(5,19)Inhibition of TNF-α and IL-1β observed in healthy subjects in this studyalign with inhibition of the same cytokine biomarkers demonstrated inanimal models for rheumatoid arthritis and cryopyrin-associated periodicsyndromes.⁵

The PD responses in this study have been expressed as ratios of plasmaconcentrations of ATI-450 to the IC₈₀ for each cytokine. Thismeasurement assists in identification of the key biomarkers that aremodulated in response to ATI-450 and allows for adjustment of ATI-450dosing to maintain plasma concentrations that are above the IC₈₀ forthose biomarkers. ATI-450 demonstrated potent inhibition of 4 of the 5biomarkers (p-HSP27, TNF-α, IL-1β, and IL-8). This inhibition wasobserved even at trough levels, as demonstrated in the 50 mg BID cohort,in which systemic drug concentrations in excess of the IC₈₀ wereachieved for p-HSP27, TNF-α, IL-1β, and IL-8 at C_(max) (3.5× to 6.0×)and C_(trough) (1.4× to 2.4×). The more modest inhibition by ATI-450 ofIL-6 induction in healthy subjects observed in this study may not beindicative of response in patients with rheumatoid arthritis because itdoes not take into account the impact of inhibiting IL-1β on downstreamIL-6 release. Studies evaluating the potency of ATI-450 in human wholeblood stimulated with IL-1β demonstrated potency for inhibiting IL-6that was comparable to that for TNF-α and IL-8 (data not shown).

ATI-450 was well tolerated at the doses investigated, exhibited dose-and time-independent (i.e., linear) PK, and dose-related PD effects.Results of this study support the progression of ATI-450 into Phase 2development for the treatment of rheumatoid arthritis and otherimmune-mediated inflammatory diseases.

TABLE 4 Summary of Demographic Characteristics Single Ascending DoseGroup ATI-450 Placebo 10 mg 30 mg 50 mg 100 mg Overall (n = 8) (n = 6)(n = 6) (n = 6) (n = 6) (N = 32) Sex, n (%) Female 6 (75.0) 5 (83.3) 6(100.0) 5 (83.3) 5 (83.3) 27 (84.4) Male 2 (25.0) 1 (16.7) 0 1 (16.7) 1(16.7)  5 (15.6) Race, n (%) White 3 (37.5) 2 (33.3) 3 (50.0)  4 (66.7)3 (50.0) 15 (46.9) Black 4 (50.0) 3 (50.0) 3 (50.0)  1 (16.7) 3 (50.0)14 (43.8) Other 1 (12.5) 1 (16.7) 0 1 (16.7) 0 3 (9.4) Ethnicity, n (%)Hispanic or 0 1 (16.7) 0 2 (33.3) 1 (16.7)  4 (12.5) Latino Not Hispanicor  8 (100.0) 5 (83.3) 6 (100.0) 4 (66.7) 5 (83.3) 28 (87.5) Latino Age,y, 37.0 ± 7.5 32.2 ± 13.7 28.7 ± 8.8 25.2 ± 4.0 31.2 ± 3.7 31.2 ± 8.8 mean ± SD Weight, kg, 76.4 ± 9.9 75.23 ± 16.78 72.8 ± 8.4 71.3 ± 8.261.5 ± 6.2 71.8 ± 11.2 mean ± SD Height, cm, 166.1 ± 5.9  172.5 ± 10.5 159.5 ± 10.0  163.8 ± 6.9  165.0 ± 8.6  165.4 ± 8.9  mean ± SD BMI,kg/m², 27.6 ± 2.6 25.1 ± 4.2  28.6 ± 2.1 26.6 ± 2.3 22.6 ± 1.1 26.2 ±3.3  mean ± SD Multiple Ascending Dose Group ATI-450 Placebo 10 mg BID30 mg BID 50 mg BID Overall (n = 6) (n = 8) (n = 8) (n = 8) (N = 30)Sex, n (%) Female 3 (50.0) 5 (62.5) 4 (50.0) 5 (62.5) 17 (56.7) Male 3(50.0) 3 (37.5) 4 (50.0) 3 (37.5) 13 (43.3) Race, n (%) White 4 (66.7) 3(37.5) 6 (75.0) 2 (25.0) 15 (50.0) Black 2 (33.3) 4 (50.0) 2 (25.0) 5(62.5) 13 (43.3) Other 0 1 (12.5) 0 1 (12.5) 2 (6.7) Ethnicity, n (%)Hispanic or 0 0 2 (25.0) 0 2 (6.7) Latino Not Hispanic or  6 (100.0)  8(100.0) 6 (75.0)  8 (100.0) 28 (93.3) Latino Age, y, 35.2 ± 6.9 36.3 ±7.8 37.9 ± 8.0 37.9 ± 9.0 36.9 ± 7.7  mean ± SD Weight, kg,  79.7 ± 17.4 72.7 ± 10.7  77.2 ± 14.4 75.9 ± 9.1 76.1 ± 12.5 mean ± SD Height, cm,170.7 ± 7.2  166.6 ± 7.1  168.1 ± 9.6  170.3 ± 10.5 168.8 ± 8.5  mean ±SD BMI, kg/m², 27.1 ± 4.4 26.2 ± 3.9 27.1 ± 2.5 26.3 ± 3.7 26.7 ± 3.5 mean ± SD BID = twice daily; BMI = body mass index.

TABLE 5 Most Common Treatment-Emergent Adverse Events Occurring in 2 orMore Subjects (Safety Set) ATI-450, n (%) Placebo, n (%) Event (n = 48)(n = 14) Dizziness 6 (12.5) 0 Headache 10 (20.8)  2 (14.3) Upperrespiratory tract 3 (6.3) 1 (7.1) infection Constipation 3 (6.3) 1 (7.1)Nausea 2 (4.2) 1 (7.1) Abdominal pain 2 (4.2) 0 Vomiting 0  2 (14.3)

TABLE 6 Pharmacokinetic Parameters of ATI-450 Single Ascending DoseGroup Multiple Ascending Dose Group ATI-450 ATI-450 ATI-450 ATI-450ATI-450 ATI-450 ATI-450 10 mg 30 mg 50 mg 100 mg 10 mg BID 30 mg BID 50mg BID Parameter (N = 6) (N = 6)^(a) (N = 6) (N = 6) (N = 8) (N = 8) (N= 8) C_(max), ng/mL 39.4 ± 10.4 122.0 ± 33.4  160.7 ± 20.4  426.0 ±110.6 51.8 ± 15.8 146.5 ± 33.6  219.0 ± 77.8  T_(max), h^(b) 2.0(2.0-4.0) 4.0 (2.0-4.1) 3.0 (2.0-4.0) 2.0 (2.0-4.0) 2.0 (2.0-2.0) 2.0(1.0-4.0) 3.0 (1.0-4.0) AUC_(0-t), h*ng/mL 276.3 ± 77.8  1074.0 ±243.5^(a) 1430.0 ± 254.0 3489.8 ± 475.7 364.6 ± 110.7 1204.6 ± 309.1 2260.3 ± 1074.7 AUC_(0-inf), h*ng/mL 285.3 ± 78.4  1102.4 ± 247.4^(a)1464.7 ± 269.5 3654.8 ± 522.9 N/A N/A N/A AUC_(tau), h*ng/mL N/A N/A N/AN/A 287.8 ± 81.5  908.9 ± 163.1 1507.8 ± 659.9  t_(1/2), h 8.5 ± 3.210.7 ± 3.3^(a)  9.1 ± 2.4 11.2 ± 5.8 9.6 ± 2.1 10.3 ± 3.2  11.6 ± 3.7 CL/F or CL_(ss)/F,^(c) L/h 37.0 ± 8.8  28.4 ± 7.1^(a) 35.1 ± 6.3 27.9 ±4.3 37.4 ± 10.9 33.9 ± 5.9  37.3 ± 11.7 V_(z)/F or V_(ss)/F,^(c) L 430.2± 124.0  451.4 ± 201.5^(a) 445.0 ± 94.7  439.8 ± 189.7 517.4 ± 203.2483.1 ± 85.1  586.4 ± 163.2 C_(trough), ng/mL^(d) Day 2 N/A N/A N/A N/A10.1 ± 4.7  37.5 ± 18.8 84.0 ± 40.6 Day 3 N/A N/A N/A N/A 9.8 ± 4.5 44.1± 29.3 80.6 ± 44.2 Day 4 N/A N/A N/A N/A 9.2 ± 5.0 36.8 ± 14.9 81.5 ±49.6 Day 5 N/A N/A N/A N/A 9.6 ± 4.7 34.9 ± 13.8 81.5 ± 43.9 Day 6 N/AN/A N/A N/A 10.7 ± 4.9  38.9 ± 16.2 76.4 ± 38.6 Day 7 N/A N/A N/A N/A11.8 ± 5.2  41.1 ± 18.3 87.9 ± 49.4 C_(min), ng/mL N/A N/A N/A N/A 8.1 ±3.1 28.8 ± 10.7 58.7 ± 32.8 C_(avg), ng/mL N/A N/A N/A N/A 24.0 ± 6.8 75.6 ± 13.4 125.6 ± 55.0  R_(ac), C_(max) N/A N/A N/A N/A 1.57 ± 0.621.17 ± 0.26 1.25 ± 0.48 R_(ac), AUC_(tau) N/A N/A N/A N/A 1.44 ± 0.321.36 ± 0.33 1.29 ± 0.37 Data presented are means ± SD unless otherwiseindicated. ^(a)One subject was excluded for AUC_(0-t), AUC_(0-inf),t_(1/2), CL/F, and V_(z)/F measurements due to withdrawn consent. Nosamples were collected past 12 hours. ^(b)Data presented are medians(ranges). ^(c)Values for CL/F and Vz/F are for single doses and valuesfor CL_(ss)/F and V_(ss)/F are for multiple doses. ^(d)C_(trough) valuesare presented for day 2 through day 7 for the MAD cohorts. AUC_(0-inf),area under the plasma concentration-time curve from time 0 to timeinfinity; AUC_(0-t), area under the plasma concentration-time curve fromtime 0 to time t; AUC_(tau), area under the plasma concentration-timecurve over the 12-hour dosing interval, tau; BID, twice daily; CL/F,clearance; CL_(ss)/F, clearance at steady state; C_(avg), average plasmaconcentration after multiple dosing, on day 7; C_(max), maximum plasmaconcentration; C_(min), minimum plasma concentration; C_(trough),measured concentration at the end of a dosing interval; N/A, notapplicable; R_(ac), AUC_(tau), accumulation ratio for AUC_(tau); R_(ac),C_(max), accumulation ratio for C_(max); t_(1/2), terminal eliminationhalf-life; T_(max), time to maximum plasma concentration; V_(ss)/F,volume of distribution at steady state; V_(z)/F, volume of distribution.

TABLE 7 Cytokine and Biomarker IC₈₀ Values and Multiples Across theDosing Interval in the 50 mg BID Dose Cohort IC₈₀ ^(a) C_(trough) ^(b)C_(max) ^(b) Biomarker (ng/mL) (Multiple of IC₈₀) (Multiple of IC₈₀)p-HSP27 36.7 2.4x 6.0x TNF-α 62.6 1.4x 3.5x IL-1β 40.8 2.2x 5.4x IL-6747.8 0.1x 0.3x IL-8 38.8 2.3x 5.6x ^(a)IC₈₀ values generated fromcombined SAD and MAD ATI-450 concentration and ex vivo inhibition datausing the WinNonlin inhibitory E_(max) model 104. ^(b)Values are from 50mg BID MAD cohort on day 7; C_(trough) = 87.9 ng/mL and C_(max) = 219ng/mL. BID, twice daily; C_(max), maximum observed plasma concentration;C_(trough), measured concentration at the end of a dosing interval;IC₈₀, concentration for 80% of maximal inhibition; IL, interleukin; MAD,multiple ascending dose; p-HSP27, phosphorylated heat shock protein 27;SAD, single ascending dose; TNF-α, tumor necrosis factor α.

Methods

Study Design. PRA Health Sciences led the study at a clinical site inLenexa, Kans., and prepared the randomization scheme for all cohortswhile maintaining blinding of observers associated with the study. PRAassigned randomization codes sequentially as healthy subjects becameeligible for randomization after enrolling in the study. For each SADdose cohort, subjects were randomly assigned to ATI-450 or placebo in anoverall 6:2 ratio. For each MAD dose cohort, subjects were randomlyassigned to ATI-450 or placebo in an overall 8:2 ratio.

Single ascending dose (SAD) cohorts. Thirty-two subjects were randomlyassigned to 1 of 4 cohorts to receive ATI-450 doses of 10, 30, 50 or 100mg (n=6 per cohort) or placebo (n=2 per cohort). Each subject received asingle oral dose of ATI-450 or placebo in the morning on an emptystomach. All subjects were admitted to the clinical study facility onday −1 and stayed until 48 hours after ATI-450 or placeboadministration.

Multiple ascending dose (MAD) cohorts. Thirty subjects were randomlyassigned to 1 of 3 cohorts to receive ATI-450 doses of 10, 30, or 50 mgBID (n=8 per cohort) or placebo (n=2 per cohort). Subjects received eachdose of ATI-450 or placebo on an empty stomach for 7 days, in themorning and the evening, with the final dose administered on the morningof day 7. Subjects were admitted to the clinical facility on day −1 andremained at the site until 72 hours after the morning dose on day 7.

Subjects. Subjects were healthy males or females between 18 and 55 yearsof age, inclusive; had a body mass index between 18 and 32 kg/m2,inclusive, with a minimum body weight of 50 kg; and tested negative forHIV-1 and HIV-2 antibodies, hepatitis B surface antigen, and hepatitis Cvirus antibody at screening. Female subjects were not pregnant ornursing and agreed to use 2 effective methods of contraception ifheterosexually active and of childbearing potential.

Subjects were excluded from the study if they had a current acute orchronic disease or used medications that may have affected their safetyor other study assessments within 2 weeks of admission.

The study was conducted in accordance with the principles of theDeclaration of Helsinki and in compliance with the International Councilfor Harmonization E6 Guideline for Good Clinical Practice, and anyapplicable national and local laws and regulations. The clinical studyprotocol, informed consent forms, and amendments to all documents werereviewed and approved by Midlands Independent Review Board of OverlandPark, Kans. Informed consent was obtained from all subjects before anystudy-related procedures began.

Safety and Tolerability Assessments. Adverse events, clinical laboratorytests, vital signs, an electrocardiogram, Holter monitoring, andphysical examination results were evaluated for each subject in bothparts of the study.

Blood Sampling and Bioanalysis. Two-milliliter blood samples werecollected for PK analysis. For the SAD cohorts, blood was collectedbefore dosing and 0.5, 1, 2, 4, 6, 8, 12, 24, 36, and 48 hours afterdosing. For the MAD cohorts, blood was collected on day 1 prior to themorning dose and 0.5, 1, 2, 4, 6, 8, and 12 hours after dosing (beforethe evening dose); before the morning dose on days 2 through 6; and onday 7, before the morning dose and 0.5, 1, 2, 4, 6, 8, 12, 13, 14, 24,36, 48, and 72 hours after the morning dose.

Blood samples of 10 mL were collected for PD analysis. For the SADcohorts, blood was collected before dosing and 1, 12, and 24 hours afterdosing. For the MAD cohorts, on day 1 blood was collected before themorning dose and 4 and 12 hours after the morning dose (prior to theevening dose); on day 7, blood was collected before the morning dose and4 and 12 hours after the morning dose.

ATI-450 concentrations in plasma samples were determined by PRA HealthSciences-Bioanalytical Laboratory (Lenexa, Kans.) using a validatedultra-performance liquid chromatography with tandem mass spectrometrymethod (details in Supplementary Information). The lower limit ofquantification was 0.500 ng/mL.

PD bioanalysis (performed by Confluence Discovery Technologies, Inc.[St. Louis, Mo.]) of phosphorylated heat shock protein 27 (p-HSP27),IL-1β, IL-6, IL-8, and TNF-α levels in blood samples were analyzed under3 conditions: (1) unstimulated, (2) ex vivo lipopolysaccharidestimulation, and (3) ex vivo lipopolysaccharide stimulation in thepresence of exogenously added 10 μM ATI-450. Historical data show thepercentage of maximum inhibition by ATI-450 achieved for a given analyteis consistent and therefore useful for normalization across samples.Additional details of the assays for analyzing p-HSP27 and cytokines areincluded in the Supplementary Information.

SAD cohort data were calculated for each subject set (day 1-pre-dosing,day 1-1 hour after dosing, day 1-12 hours after dosing, and day 2-24hours after dosing) as the % of day 1 pre-dosing using the ex vivolipopolysaccharide stimulation as the maximum signal and normalizingacross the sample set using the day 1 ex vivo lipopolysaccharide plus 10μM ATI-450 as maximum inhibition.

MAD cohort data were calculated for each subject set (day 1—pre-dosing,day 1-4 hours after dosing, day 1-12 hours after dosing, day7—pre-dosing, day 7-4 hours after dosing, and day 7-12 hours afterdosing) as the % of day 1 pre-dosing using the ex vivolipopolysaccharide stimulation as the maximum signal and normalizingacross the sample set using the day 1 ex vivo lipopolysaccharide plus 10μM ATI-450 as maximum inhibition.

Pharmacokinetics. ATI-450 plasma concentrations and PK parameters weredetermined for all SAD and MAD cohorts. Noncompartmental analysis withPhoenix WinNonlin™, version 8.1 (Certara, Princeton, N.J.) was used todetermine the following PK parameters from the plasma concentration-timedata for ATI-450: maximum observed plasma concentration (Cmax); time tomaximum observed plasma concentration (T_(max)); area under the plasmaconcentration-time curve from time 0 through time t (AUC_(0−t)), where tis the time of the last quantifiable concentration; area under theplasma concentration-time curve from time 0 to infinity (AUC_(0−inf));apparent terminal elimination rate constant (λz) and associatedhalf-life (t_(1/2)); apparent clearance (C_(L/F)); and apparent volumeof distribution (V_(z/F)). For the MAD cohorts, several additional PKparameters were determined, including area under the plasmaconcentration-time curve over the 12-hour dosing interval, tau(AUC_(tau)); minimum observed plasma concentration in the dosinginterval after multiple dosing, on day 7 (C_(min)); average plasmaconcentration after multiple dosing, on day 7 (C_(avg)); measuredconcentration at the end of a dosing interval (C_(trough)); accumulationratio for C_(max) and AUC_(tau) (R_(ac),C_(max) and R_(ac),AUC_(tau));apparent clearance at steady state (CL_(ss/F)); and apparent volume ofdistribution at steady state (V_(ss/F)).

Pharmacodynamics. PD data analysis was conducted using Microsoft Excel2010 (Microsoft, Redmond, Wash.), Meso Scale Discovery Workbench®analysis software (Rockville, Md.), and Phoenix WinNonlin, version 8.2.For the PK/PD analysis in Phoenix WinNonlin, the ATI-450 plasmaconcentrations and the percentages of inhibition of production oflipopolysaccharide-stimulated p-HSP27, TNF-α, IL-1β, IL-6 and IL-8levels in human whole blood from all SAD/MAD cohorts were simultaneouslyfit using an inhibitory E_(max) model (WinNonlin, model 104). Theparameters estimated by the model for each biomarker were the baselineeffect at zero concentration (E₀), the maximum percent inhibition(Imax), and the ATI-450 plasma concentration at half maximal inhibition(IC₅₀). The ATI-450 concentrations at 80% inhibition (IC₈₀) weresubsequently calculated from the IC₅₀ estimates using the followingequation: IC_(x)=(x/100−x)*IC₅₀, where x is the percent inhibition(e.g., 80%). Additionally, multiples of the peak and troughconcentrations of ATI-450 at 50 mg BID on day 7 to the IC₅₀ and IC₈₀estimates for p HSP27 and each cytokine were determined.

Sample size calculations. No prospective calculations of statisticalpower were made for sample size. Sample sizes of 32 subjects and 30subjects for the SAD and MAD cohorts, respectively, were selected andare typical for a first-in-human study.

Bioanalysis Assay of ATI-450. Sample processing was performed by proteinprecipitation using a 50.0-μL sample volume. Separation betweenpotential metabolites and interfering endogenous compounds was achievedby liquid chromatography with tandem mass spectrometry using a WatersAcquity UPLC BEH C18 column (2.1 ×50 mm, 1.7 μm particle size) at 40° C.using 10 mM ammonium formate with 0.3% formic acid as mobile phase A and50:50:0.3 acetonitrile:methanol:formic acid as mobile phase B, operatingat isocratic conditions (with post-elution gradient wash-off) with aflow rate of 0.700 mL/min. A triple quadrupole 6500 mass spectrometerequipped with a turbo-ion spray source was used for detection inpositive ion mode. Quantification was based on multiple reactionmonitoring of the transitions of m/z 514.3→297.2 for ATI-450 and517.3→336.2 for the internal standard, ATI-450-¹³C₃. A linearcalibration curve ranging from 0.500 to 500 ng/mL with a 1/x² weightingfactor was used. The method was minimally required to have intra- andinter-day precision (coefficients of variation) for pooled plasmaquality control samples of ≤15% except at the lower limit ofquantitation, where ≤20% was acceptable. The calculated concentrations(both inter- and intra-day) were required to be within 15% of nominal atall concentrations except the lower limit of quantitation, where up to20% deviation from nominal was acceptable. The precision and accuracy ofthe method exceeded these minimum requirements for assay validation. Inaddition, stability of the analyte in frozen K₂-EDTA human plasma wasdemonstrated for periods exceeding the storage periods of the samplesprior to analysis, as well as under all conditions to which studysamples or working solutions were subjected.

Sample Preparation for p-HSP27 Analysis. For p-HSP27 analysis, 1-mLaliquots of each sample were transferred into six 2-mL microtubes withsubsequent treatment designation: ‘A’ for lipopolysaccharide stimulationonly (maximum signal), ‘B’ for ex vivo added ATI-450 samples thatreceived lipopolysaccharide stimulation (maximum inhibition), ‘C’ forunstimulated. Treatment groups consisted of 3 tubes for group ‘A’, 2tubes for group ‘B’, and 1 tube for group ‘C’.

Dimethyl sulfoxide (DMSO) was added to all tubes in groups ‘A’ and ‘C’to a final concentration of 0.1% (10 μL per mL of a 10% DMSO solution inDulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovineserum (FBS) and 1% penicillin/streptomycin/glutamine). Samples were thenrocked gently at room temperature. A stock solution of ATI-450 at 10 mMin 100% DMSO was diluted to 1 mM in DMEM containing 10% FBS and 1%penicillin/streptomycin, and 10 μL of this working solution was addedper mL of blood in group ‘B’ samples to give a final concentration of 10μM in 0.1% DMSO. Samples were then rocked gently at room temperature fora minimum of 1 hour.

Samples in groups ‘A’ and ‘B’ were stimulated with lipopolysaccharide ata final concentration of 100 ng/mL for 22 minutes at room temperature. Astock solution of lipopolysaccharide at 1 mg/mL in DMEM containing 10%FBS and 1% penicillin/streptomycin was diluted to 10 μg/mL in DMEMcontaining 10% FBS and 1% penicillin/streptomycin, and 10 μL of thisworking solution was added per mL of blood. Samples were gently rockedat room temperature for 11 minutes, then placed upright for 11 minutesprior to peripheral blood mononuclear cells (PBMC) isolation.

Ten μL of DMEM containing 10% FBS only was added per mL of blood ingroup ‘C’ for all patient samples (unstimulated samples). Samples weregently rocked at room temperature for 11 minutes, then placed uprightfor 11 minutes prior to PBMC isolation.

Upon completion of treatment (lipopolysaccharide stimulated orunstimulated) for each sample, PBMCs were isolated from each human wholeblood aliquot in all treatment groups. One mL of blood from each samplewas gently layered onto 0.75 mL of Histopaque-1077 in a 2-mLmicrocentrifuge tube. Histopaque-1077 was maintained at roomtemperature. The samples were centrifuged for 2 minutes at 16,000×g inan Eppendorf microcentrifuge. The interface and upper layers wereremoved and added to tubes containing 1 mL cold Dulbecco'sphosphate-buffered saline (DPBS). These samples were then centrifugedfor 30 seconds at 16,000×g in an Eppendorf microcentrifuge to pellet thecells. The buffer supernatant was removed by aspiration and the pelletswere re-suspended in 1 mL of cold DPBS. The pellets from each samplewere then re-pelleted, as described above. The buffer was removed byaspiration and the final pellets were lysed in 100 μL of complete lysisbuffer (MSD Tris lysis buffer, 1× Halt™ Protease inhibitor cocktail, 1×phosphatase inhibitor cocktail 2, 1× phosphatase inhibitor cocktail 3, 2mM PMSF, 2 mM sodium fluoride and 1 mM sodium vanadate). Samples werevortexed until the cell pellet fully dissolved and then were flashfrozen on dry ice.

Analysis of p-HSP27 (Ser78) Levels Using Luminex Technology. The lysateswere analyzed for HSP27 phosphorylation with a custom Milliplex® MAPHuman Phospho-HSP27(Ser78) Cell Signaling Magnetic Bead Kit fromMillipore Sigma (Burlington, Mass.) using the kit-prescribed immunoassayprotocol for a 96-well solid plate and hand-held magnetic separationblock. Once all sample lysates from a given cohort were generated, thelysates were thawed at 4° C. and transferred to a round-bottom 96-wellpolypropylene master plate. A daughter plate consisting of a 2-folddilution of each lysate (17 μL of lysate plus 17 μL of Milliplex® MAPassay buffer 2) was generated for each master plate. The diluted lysatesfrom the daughter plates were used in the assay.

The 96-well, flat-bottom, clear-bottom black plates provided in the kitwere prewashed with 50 μL/well of assay buffer 2 for 10 minutes. Thewash was decanted and 1× HSP27 magnetic beads in assay buffer 2 wereadded to each well at 25 μL/well. The diluted lysates from the daughterplates were then added at 25 μL/well. The plates were sealed andincubated overnight at 4° C. with shaking in the dark.

After the overnight incubation, using the hand-held magnetic separationblock, the samples were decanted and plates were washed 2 times withassay buffer 2. Biotin-labeled detection antibody (1× in assay buffer 2)was added at 25 μL/well to all plates and incubated for 1 hour. Allincubations were at room temperature with shaking in the dark. Thedetection antibody was then decanted using the hand-held magneticseparation block and streptavidin-phycoerythrin (PE; 1× in assay buffer2) was added to each well at 25 μL/well. The plates were then incubatedfor 15 minutes. Amplification buffer (1× in assay buffer 2) was added tothe streptavidin-PE-containing wells at 25 μL/well and incubated anadditional 15 minutes. Using the hand-held magnetic separation block,the streptavidin-PE/amplification buffer was removed by decanting. Assaybuffer 2 was added to each well at 150 μL/well. The plates were placedon the plate shaker for a minimum of 5 minutes, then analyzed using theLuminex° 100/200 Instrument (Luminex Corp, Austin, Tex.).

Sample Preparation for Cytokine Analysis. For each sample, 180 μLaliquots were transferred into 7 wells of a round bottom 96 well tissueculture polystyrene low evaporation plate. Of the 7 wells for eachsample, 3 were designated ‘A’ for lipopolysaccharide stimulation only(maximum signal), 2 were designated ‘B’ for ex vivo added ATI-450samples that received lipopolysaccharide stimulation (maximuminhibition), and 2 were designated ‘C’ for unstimulatedlipopolysaccharide. No outer wells on the plates were used and all wellssurrounding the samples contained 200 μL of phosphate buffered saline.

DMSO was added to all wells in groups ‘A’ and ‘C’ to a finalconcentration of 0.1% (10 L/well of a 2% DMSO solution in DMEMcontaining 10% FBS and 1% penicillin/streptomycin/glutamine). A stocksolution of ATI-450 at 10 mM in 100% DMSO was diluted to 200 mM in DMEMcontaining 10% FBS and 1% penicillin/streptomycin, and 10 mL of thisworking solution was added per well to all wells in group ‘B’ to give afinal concentration of 10 mM in 0.1% DMSO. Samples were mixed gently for30 seconds on a plate shaker using pin tools, then placed in anincubator at 37° C./5% CO₂ for 1 hour.

Samples in groups ‘A’ and ‘B’ were stimulated with lipopolysaccharide ata final concentration of 100 ng/mL. A stock solution oflipopolysaccharide at 1 mg/mL in DMEM containing 10% FBS and 1%penicillin/streptomycin was diluted to 2 mg/mL in DMEM containing 10%FBS and 1% penicillin/streptomycin, and 10 mL of this working solutionwas added per well of blood for all samples in groups ‘A’ and ‘B’. TenmL of DMEM containing 10% FBS only was added per well of blood for allsamples in group ‘C’ (unstimulated samples). Samples were mixed gentlyfor 30 seconds on a plate shaker using pin tools, then placed in anincubator at 37° C./5% CO₂ for 5 hours.

Following the incubation, the plates were removed from the incubator andsealed with adhesive clear plate seals. The plates were then centrifugedfor 10 minutes at 1800×g at room temperature. Seventy-five mL of plasmawas removed from each well and transferred to a 96-well polypropyleneround bottom master plate, which was then sealed and frozen at −80° C.until all samples from the respective cohort were generated. Once allsamples from a given cohort were generated, the samples were analyzedfor cytokine (TNF-α, IL-1b, IL-6, and IL-8) production using Meso ScaleDiscovery Technology.

Analysis of Cytokine Levels Using Meso Scale Discovery Technology. Alllipopolysaccharide plasma samples were analyzed for cytokine productionwith a V-Plex Human Proinflammatory Panel II (4-Plex) Kit from MesoScale Discovery (Rockville, Md.). The master plates containing theplasma samples were thawed on ice, and a daughter plate consisting of a25-fold dilution of each plasma sample (3 μL of plasma plus 72 μL ofMeso Scale Discovery Diluent 2) was generated for each master plate. Thediluted plasma samples from the daughter plates were used in thecytokine analysis. The Meso Scale Discovery 4-plex plates were washed 3times with 150 μL/well of wash buffer (1× KPL phosphate buffered salinewith Tween 20 from SeraCare [Milford, Mass.]). Prepared calibrators andthe samples from the daughter plates were added to the washed Meso ScaleDiscovery 4-plex plates at 50 μL/well. The 4-plex plates were sealed andshaken overnight at 4° C.

After the overnight incubation, the samples were removed by decanting.The 4-plex plates were then washed 3 times with 150 μL/well of washbuffer. A detection antibody mix consisting of sulfo-tagged anti-humanIL-1β, sulfo-tagged anti-human IL-6, sulfo-tagged anti-human IL-8, andsulfo-tagged anti-human TNF-α antibodies in Meso Scale Discovery Diluent3 was added to each well at 25 μL/well, and the plates were sealed andincubated for 2 hours at room temperature with shaking. Following theincubation, the detection antibodies were removed by decanting. The4-plex plates were washed 3 times with 150 μL/well of wash buffer afterwhich 2× Read Buffer was added at 150 μL/well to all wells. The plateswere then read using a Meso Scale Discovery Sector S 600 instrument.Calculations to establish cytokine calibration curves and to determineanalyte concentrations in the samples were carried out using the MesoScale Discovery Workbench analysis software.

TABLE 8 Statistical Analysis of Dose Proportionality of ATI-450 in SADCohorts 90% CI of PK Parameter n Intercept Slope Slope C_(max) (ng/mL)24 1.302 1.006 (0.902, 1.111) AUC_(0-t) (h*ng/mL) 23 3.113 1.088 (0.997,1.180) AUC_(0-inf) 23 3.134 1.091 (0.999, 1.184) (h*ng/mL)The dose proportionality analyses were performed using the power model:In(PK)=intercept+slope*In(dose)+e, where PK is the PK parameter and e isthe error term. A value of slope=1 indicates dose proportionality. InTable 8, the following terms are used: AUC, area under theconcentration-time curve; C_(max), maximal plasma concentration; CI,confidence interval; n, number of observations; PK, pharmacokinetic.

TABLE 9 Statistical Analysis of Dose Proportionality of ATI-450 in MADCohorts 90% CI of PK Parameter n Intercept Slope Slope C_(max) (ng/mL)24 1.857 0.899 (0.757, 1.042) AUC_(tau) (h*ng/mL) 24 3.292 1.019 (0.872,1.167)The dose proportionality analyses were performed using the power model:In(PK)=intercept+slope*In(dose)+e, where PK is the PK parameter and e isthe error term. A value of slope=1 indicates dose proportionality. InTable 9, the following terms are used: AUC, area under theconcentration-time curve; C_(max), maximal plasma concentration; CI,confidence interval; n, number of observations; PK, pharmacokinetic.

TABLE 10 Inhibitory E_(max) Model Parameters P-HSP27 TNF-α IL-1β IL-6IL-8 IC₅₀, ng/mL 9.2 15.6 10.2 186.9 9.7 IC₈₀, ng/mL 36.7 62.6 40.8747.8 38.8 I_(max), % 110.6 96.5 74.3 116.4 57.1In Table 10, the following terms are used: IC₅₀, plasma concentration athalf maximal inhibition; IC₈₀, plasma concentration at 80% of maximalinhibition; IL, interleukin; I_(max), maximum percent inhibition;p-HSP27, phosphorylated heat shock protein 27; TNF, tumor necrosisfactor.

Example 2 Extension Study of Higher Doses of Orally Administered ATI-450

This extension of the Phase I clinical study described in Example 1 wascarried out to establish the safety and tolerability of higher doses (80mg and 120 mg twice daily) of ATI-450 in healthy volunteers.

Methods: Safety, pharmacokinetics (PK) and pharmacodynamics (PD) wereassessed in two cohorts of subjects in a randomized, observer-blind,placebo-controlled study in male and female healthy subjects aged 18-55(n=77). The study consisted of twenty (20) subjects enrolled into 2cohorts. At each dose level/cohort a total of 10 subjects wererandomized to receive multiple oral doses of ATI-450, i.e., 80 mg or 120mg (n=8), or placebo (n=2) twice daily (BID) for 7 days. The final dosewas administered in the morning on Day 7. Blood samples were obtainedDay 1—before dosing, Day 1—4 hours after dosing and Day 1—12 hours afterdosing and Day 7—before dosing, Day 7—4 hours after dosing, day 7—12hour after dosing and Day 7—24 hours after dosing for each subject.

Results. The graphs of FIG. 4A-4E show the continued dose-dependentinhibition of LPS-stimulated pHSP27 (FIG. 4A, top), TNF-α (FIG. 4B,top), IL-1β (FIG. 4C, top), IL-8 (FIG. 4D, top), IL-6 (FIG. 4E, top)from Day 7 blood samples taken from subjects dosed with 10 mg, 30 mg, 50mg, 80 mg, and 120 mg of ATI-450. FIGS. 4A-4E also show mean (±SEM)levels of pHSP27 (FIG. 4A, bottom), TNF-α (FIG. 4B, bottom), IL-1β (FIG.4C, bottom), IL-8 (FIG. 4D, bottom), IL-6 (FIG. 4E, bottom) in subjectsadministered 10 mg, 30 mg, 50 mg, 80 mg, and 120 mg twice daily,comparing day 1 pre-dosing values (set to 100%) with day 7 values 4hours after dosing (approximate C_(max)) and 12 hours after dosing(C_(trough)). These results extend the findings of Example 1 to showthat higher doses of ATI-450 have increased benefit in the inhibition ofpro-inflammatory cytokines.

ATI-450 at the higher doses does not inhibit the regulatoryanti-inflammatory cytokine IL-10 to the same extent as pro-inflammatorycytokines. FIG. 5 is a graph showing differential modulation of ex vivostimulated IL-10 versus TNF-α and IL-1β in blood samples taken fromsubjects dosed with placebo or 80 mg or 120 mg of ATI-450. IL-10 wasonly modulated 25-30% at doses of drug that generated near maximalinhibition of proinflammatory cytokines (TNF-α and IL-1β).

ATI-450 potently inhibits ex vivo IL-1β-induced pro-inflammatorycytokines TNF-α, IL-6, and IL-8 whether stimulated by LPS or IL-1β. FIG.6 depicts three graphs comparing ATI-450 modulation of LPS and IL-1βstimulated TNF-α (far left), IL-6 (middle), and IL-8 (far right)production.

FIGS. 7A-7C and Table 8 below summarize the pharmacokinetic data ofATI-450 following 80 mg and 120 mg BID dosing. FIG. 7A depict graphsshowing mean plasma concentration-time profiles of ATI-450 after 1 day(top) and 7 days (middle and bottom) of BID dosing, semi-log scale. BID,twice daily. FIG. 7B shows mean (±std. dev.) plasma concentration-timeprofiles of ATI-450 dosed at 120 mg BID. FIG. 7C depicts graphs showingthe plasma concentration-time profiles of ATI-450 in individual subjectsat days 1 (left) and 7 (right) following 120 mg BID dosing.

A summary of the mean pharmacokinetic parameters of ATI-450 80 mg and120 mg BID dosing is provided in Table 11 below. The correspondingparameters for the 50 mg BID cohorts is included for comparison.

TABLE 11 Pharmacokinetic Parameters of ATI-450 80 mg and 120 mg BIDdosing Day 1 Day 7 ATI-450 ATI-450 ATI-450 ATI-450 ATI-450 ATI-450 50 mg80 mg 120 mg 50 mg BID 80 mg BID 120 mg BID Parameter (N = 8) (N = 8) (N= 8) (N = 8) (N = 8) (N = 8) C_(max), ng/mL 186.7 ± 66.8 258.4 ± 83.8378.9 ± 60.6 219.0 ± 77.8   388.8 ± 100.8 416.5 ± 122.9 t_(max), h^(a)2.0 (2.0-4.0) 2.0 (1.0-4.0) 3.0 (2.0-3.0) 3.0 (1.0-4.0) 2.0 (2.0-3.0)2.0 (1.0-4.0) AUC_(0-t), h*ng/mL 1179.5 ± 340.1 1679.2 ± 451.2 2314.5 ±379.9 2260.3 ± 1074.7  4031.3 ± 1152.5 4171.0 ± 2094.4 AUC_(tau),h*ng/mL 1179.5 ± 340.1 1685.7 ± 453.5 2322.1 ± 383.2 1507.8 ± 659.9 2548.0 ± 729.9 2577.8 ± 1030.9 t_(1/2), h N/A N/A N/A 11.6 ± 3.7  14.3 ±5.3 8.7 ± 2.4 CL_(ss)/F, L/h N/A N/A N/A 37.3 ± 11.7 33.2 ± 7.6 52.3 ±17.4 V_(ss)/F, L N/A N/A N/A 586.4 ± 163.2  690.4 ± 293.8 621.3 ± 182.8C_(trough), ng/mL^(b) Day 2 N/A N/A N/A 84.0 ± 40.6 175.5 ± 67.5 152.5 ±93.3  Day 3 N/A N/A N/A 80.6 ± 44.2 148.6 ± 54.0 144.4 ± 98.8  Day 4 N/AN/A N/A 81.5 ± 49.6 147.9 ± 59.9 126.0 ± 84.4  Day 5 N/A N/A N/A 81.5 ±43.9 144.7 ± 78.3 145.3 ± 67.5  Day 6 N/A N/A N/A 76.4 ± 38.6 144.5 ±64.9 160.8 ± 87.5  Day 7 N/A N/A N/A 87.9 ± 49.4 158.3 ± 60.3 155.8 ±93.1  C_(min), ng/mL N/A N/A N/A 58.7 ± 32.8 100.5 ± 39.8 96.1 ± 64.1C_(avg), ng/mL N/A N/A N/A 125.6 ± 55.0  212.3 ± 60.8 214.8 ± 85.9 R_(ac), AUC_(tau) N/A N/A N/A 1.29 ± 0.37  1.53 ± 0.22 1.08 ± 0.26 Datapresented are means ± SD unless otherwise indicatec. ^(a)Data presentedare medians (ranges). ^(b)C_(trough) values are presented for day 2through day 7 for the MAD cohorts. AUC_(0-t), area under the plasmaconcentration-time curve from time 0 to time t; AUC_(tau), area underthe plasma concentration-time curve over the 12-hour dosing interval,tau; BID, twice daily; CL_(ss)/F, clearance at steady state; C_(avg),average plasma concentration after multiple dosing, on day 7; C_(max),maximum plasma concentration; C_(min), minimum plasma concentration;C_(trough), measured concentration at the end of a dosing interval; N/A,not applicable; R_(ac), AUC_(tau), accumulation ratio for AUC_(tau);t_(1/2), terminal elimination half-life; T_(max), time to maximum plasmaconcentration; V_(ss)/F, volume of distribution at steady state.

Quantitation of changes in analyte concentration relative to ATI-450dose in the five MAD cohorts is summarized in Table 12 and FIGS. 4A-4E.

TABLE 12 Bio- Dose Level IC₅₀ IC₈₀ IC₉₀ marker (mg BID) C_(trough)C_(max) C_(trough) C_(max) C_(trough) C_(max) IL-1β 10 0.7 3.2 0.2 0.80.1 0.4 IL-6 10 0.1 0.4 0.0 0.1 0.0 0.0 IL-8 10 0.5 2.3 0.1 0.6 0.1 0.3pHSP27 10 1.3 5.8 0.3 1.4 0.1 0.6 TNF-α 10 0.7 3.1 0.2 0.8 0.1 0.3 IL-1β30 2.6 9.2 0.6 2.3 0.3 1.0 IL-6 30 0.3 1.2 0.1 0.3 0.0 0.1 IL-8 30 1.86.4 0.5 1.6 0.2 0.7 pHSP27 30 4.6 16.3 1.1 4.1 0.5 1.8 TNF-α 30 2.5 8.80.6 2.2 0.3 1.0 IL-1β 50 5.5 13.7 1.4 3.4 0.6 1.5 IL-6 50 0.7 1.7 0.20.4 0.1 0.2 IL-8 50 3.8 9.6 1.0 2.4 0.4 1.1 pHSP27 50 9.8 24.3 2.5 6.11.1 2.7 TNF-α 50 5.3 13.1 1.3 3.3 0.6 1.5 IL-1β 80 9.9 24.3 2.5 6.1 1.12.7 IL-6 80 1.2 3.1 0.3 0.8 0.1 0.3 IL-8 80 6.9 17.0 1.7 4.3 0.8 1.9pHSP27 80 17.6 43.2 4.4 10.9 2.0 4.8 TNF-α 80 9.5 23.3 2.4 5.8 1.1 2.6IL-1β 120 9.7 26.0 2.4 6.5 1.1 2.9 IL-6 120 1.2 3.3 0.3 0.8 0.1 0.4 IL-8120 6.8 18.2 1.7 4.6 0.8 2.0 pHSP27 120 17.3 46.3 4.3 11.6 1.9 5.2 TNF-α120 9.3 24.9 2.3 6.2 1.0 2.8

The data in Table 12 summarizes the inhibitory concentrations of pHSP27and the four cytokines by ATI-450 relative to the C_(trough) and C_(max)in the 5 MAD dose cohorts (10 mg, 30 mg, 50 mg, 80 mg and 120 mg). Thedata is expressed as the ratio of ATI-450 concentrations at C_(max) andC_(trough) within the various dose cohorts relative to the IC₅₀, IC₈₀and IC₉₀ estimates for each analyte. The respective mean C_(trough) andC_(max) values of ATI-450 on day 7 were 11.83 and 51.8 ng/mL at 10mgBID, 41.14 and 146.5 ng/mL at 30mg BID,87.94 and 219.0 ng/mL at 50 mgBID, 158.25 and 388.8 ng/mL at 80 mg BID, and 155.81 and 416.5 ng/mL at120 mg BID. A ratio of 1.0 indicates a concentration that is equivalentto the IC value being compared. Ratios that are less than 1 are shown innormal type-face, ratios that are ≥1 are bolded. For the 50 mg BID dosecohort, concentrations at C_(trough) were similar to or in excess of therelative IC₈₀ concentrations (1.0 to 2.5-fold) for the target biomarkerpHSP27 and three of the four cytokines (TNF-α, IL-1β, and IL-8) but notfor IL-6. Trough concentrations from the 80 mg BID and 120 mg BID dosesproduced larger, but comparable, multiples of the IC₈₀ concentrationsfor pHSP27, TNF-α, IL-1β, and IL-8 (1.7- to 4.4-fold and 1.7- to4.3-fold, respectively) relative to 50 mg BID. The effect of ATI-450dose on the relative concentration of each cytokine analyte in the exvivo stimulated assay expressed as a percentage of pre-dosing analytelevels (set to 100%) for the 10 mg, 30 mg and 50 mg BID MAD cohorts onDay 7, 4 hours after dosing and 12 hours after dosing is shown in FIGS.4A-4E. The 4-hour after dosing Day 7 samples were utilized to reflectapproximate steady state C_(max) ATI-450 concentrations while the12-hour after dosing Day 7 samples were used to reflect steady-stateC_(trough) concentrations of the drug. A marked dose-dependent reductionin concentration is observed for all four cytokines at the four-hourtime-point and persists through twelve hours. TNF-α, IL-1β, and IL-8 alldemonstrated a reduction in concentration that persisted for the entiredosing interval and did not appreciably change between 4 and 12 hoursafter dosing.

ATI-450 demonstrated both concentration-dependent and dose-dependentmodulation of the target biomarker pHSP27 and inhibition of theproduction of the four cytokines analyzed, TNF-α, IL-1β, IL-6, and IL-8.Concentration-response parameters for ATI-450 reduction of pHSP27,TNF-α, IL-1β, and IL-8 were comparable, while the parameters for IL-6were higher. At the 50 mg BID dose, concentrations at C_(trough)following seven days of dosing were similar to or in excess of the IC₈₀for ATI-450 reduction of pHSP27, as well as for ATI-450 inhibition ofproduction of TNF-α, IL-1β, and IL-8. Trough concentrations from the 80mg BID and 120 mg BID doses produced larger, but comparable, multiplesof the IC80 concentrations relative to 50 mg BID suggesting that agreater level of target inhibition may be achieved at these higherdoses.

The higher dosing regimens, i.e., 80 mg and 120 mg BID, were welltolerated with no severe adverse effects reported. A summary of thesafety data is provided in Table 13 below.

TABLE 13 Preliminary Safety Data in Patients Administered 80 mg or 120mg BID Cohort 1 Cohort 2 (80 mg (120 mg BID, Cohort 1 BID, Cohort 2 AE n= 8) (placebo) n = 8) (placebo) Intensity Headache 2 (25%) 1 (50%) 7(88%) 1 (50%) Mild Dizziness 2 (25%) 6 (75%) Mild Xerosis 1 (13%) 5(63%) Mild Constipation 1 (14%) Mild Nausea 2 (25%) Mild Parasthesia 2(25%) Mild Abdominal 1 (13%) Mild Pain Loose stools 1 (13%) MildPharyngitis 1 (13%) Mild # only 1^(st) or 2^(nd) day + 7 cases resolvedon drug * After stopping drug

Example 3 Non-Clinical Safety Assessment of 13-Week Oral Administrationof ATI-450

Study Design. In this study, 13-week oral administration of ATI-450 wastested in rate and mini-pigs. Rats were administered 0 (vehicle), 3, 10and 30 mg/kg/day, and Mini-pigs were administered 0 (vehicle) 10, 30 and60 mg/kg/day

Results: The no-observed adverse effect level (NOAEL) for rat was 30mg/kg/day, and for mini-pig was 60 mg/kg/day. No study drug-relatedmortality was observed.

Rat non adverse findings: minimal ulceration/mixed cell inflammation wasobserved around nasal area in several animals at mid and high dose.Minimal to moderate degeneration of myocytes at all doses, includingcontrol animals. However, complete recovery following 28-day non-dosingperiod was observed.

Mini-pig non-adverse findings: clinical observations at ≥30 mg/kg/day.Slightly increased neutrophil, lymphocyte and platelet counts wereobserved along with decreased red cell mass in males at ≥10 mg/kg/dayand females at 60 mg/kg/day.

TABLE 14 Mean Systemic Exposures (C_(max) and AUC₀₋₂₄) in Rats andMinipigs Given Daily Oral Doses of Study Drug for 13-weeks Compared toPreliminary Mean Systemic Exposures (C_(max) and AUC₀₋₁₂) in HealthyHuman Subjects Given Twice Daily Oral Doses of 50 mg, 80 mg, and 120 mgStudy Drug for 6.5 days. Exposure Multiples Study C_(max) AUC₀₋₂₄Rat/Human Minipig/Human Species Dose Day (ng/mL) (ng · h/mL) C_(max) AUCC_(max) AUC Rat 30 m g/kg/day 88 M = 3260 M = 36400 — — — —  F = 5440  F= 43500 Minipig 60 mg/kg/day 90 M = 1670 M = 14400 — — — —  F = 2360  F= 22600 Human 50 mg BID 7 219.0 3042^(a)  15-25 12-14 7.6-11  4.7-7.4 80mg BID 7 388.8 5096^(a) 8.4-14 7.1-8.5 4.3-6.1 2.8-4.4 120 mg BID 7416.5 5156^(a) 7.8-13 7.1-8.4 4.0-5.7 2.8-4.4 ^(a)Human AUC valuerepresents the total daily exposure calculated as AUC₀₋₁₂ × 2

Example 4 Oral Composition of ATI-450 for Treatment of RheumatoidArthritis

A Phase 2a, randomized, investigator and patient-blind,sponsor-unblinded, parallel group, placebo-controlled study wasconducted to investigate the safety, tolerability, PK, and PD of ATI-450plus MTX versus MTX alone in patients with moderate to severe RA.

Approximately 19 patients were enrolled with the expectation that atleast 15 patients would complete the 12-weeks of treatment. The studyconsists of an up to 28-day screening period, a 12-week treatmentperiod, and a 4-week follow-up period. The total duration of the studyfor patients remaining until their final follow-up assessment is 20weeks.

The primary objective was to evaluate the safety and tolerability ofATI-450 plus MTX in patients with moderate to severe rheumatoidarthritis (RA). The secondary objective of the study was to assess (i)the PD profiles of ATI-450 plus MTX in patients with moderate to severeRA, and (ii) the pharmacokinetics of ATI-450 in patients with moderateto severe RA who are receiving concomitant MTX. In addition to theprimary and secondary objectives, the PD profiles of ATI-450 plus MTX inpatients with moderate to severe RA was assessed.

Patient eligibility was assessed based on the following criteria: (i) adiagnosis of adult onset RA (ACR/EULAR classification criteria); (ii)DAS28-CRP≥3.2 defined as moderate to high disease activity; (iii)moderately to severely active RA defined by at least 4/28 tender and4/28 swollen joints; (iv) hsCRP≥5 mg/L at screening; (v) definitiveintra-articular synovitis or osteitis defined as a score of 1 or greateron a Hand-Wrist MM (using RAMRIS); (vi) stable MTX dose (defined as 7.5mg to 25 mg weekly) for at least 4 weeks prior to the screening visit.Patients whose eligibility was confirmed at baseline were randomized ina 3:1 ratio to receive either ATI-450 tablets (50 mg twice daily [BID])plus MTX, or matching placebo tablets plus MTX. Study medications wereadministered orally for 12 weeks. Patients were required to remain on astable dose of MTX (7.5 mg to 25 mg/week) and a stable dose of folic orfolinic acid (≥5 mg/week) for the duration of the study.

Patients attended clinic visits on Days 7, 14, 28, 42, 56, and 84 (±1day) for safety, efficacy, PK, and PD assessments. The morning dose ofstudy medication was administered in the clinic on each study visit day.

At the completion of 4 weeks of treatment (Day 28), each patient'ssafety data (e.g., AEs, laboratory values, vital signs, and ECGs) willbe reviewed to ensure that the patient is tolerating the treatmentregimen and is deemed suitable to continue treatment for the next 8weeks.

On Day 84 (Week 12), patients will complete the end of studyassessments. A safety follow-up visit will be conducted 30 days (+7)after the last dose of study medication.

Efficacy Assessments and Results

A summary of patient demographic is provided in Table 15 below.

TABLE 15 Patient Demographics Statistic/ Parameter Category PlaceboATI-450 Age (year) n 3 16 Mean (SD) 55.33 (6.807) 55.88 (9.926) Median53    59.5 Min-Max 50-63 32-65 Sex Female 3/3 (100%) 11/16 (68.75%) Male0/0 (0%) 5/16 (31.25%) Weight (kg) n 3 16 Mean (SD) 98.93 (14.616) 92.74(25.504) Median  105.4   88.15 Min-Max  82.2-109.2  52.7-141.5 BMI n 316 Mean (SD) 36.53 (4.737) 33.37 (8.829) Median  38.7   30.6 Min-Max31.1-39.8 20.6-51.7 Duration n 3 16 of Disease Mean (SD) 7.5 (11.364)9.98 (9.554) Median   1.6    6.45 Min-Max  0.3-20.6  0.3-33.4 hsCRP(mg/L) n 3 16 Median  21.3   11.7 Min-Max 12.6-31.2  2.6-29.5 DAS-28 n 316 Median   5.3    5.65 Min-Max 5.3-6.7 3.9-7.4 Mean (SD) 5.77 (0.808)5.71 (0.937) Race Black Or 1/3 (33.33%) African American Asian 1/16(6.25%) White 2/3 (66.67%) 15/16 (93.75%) Ethnicity Hispanic 4/16 (25%)Or Latino Not Hispanic 3/3 (100%) 12/16 (75%) Or Latino

Overall the patient population exhibited high disease activity asindicated by the median Disease Activity Score for 28 Joint Count(DAS-28) (see Smolen et al., “Validity and reliability of thetwenty-eight-joint count for the assessment of rheumatoid arthritisactivity.” Arthritis Rheum 38(1):38-43 (1995), which is herebyincorporated by reference in its entirety) for both the treatment group(5.65) and the placebo group (6.0). In addition, the patient populationexhibited a broad range of disease duration of 0.3-34 years, with manypatient having high hsCRP despite long history and multiple treatmentoption. This suggests a relatively refractory group of patients.

Disease Activity Score for 28 Joint Count. Assessments of RA by theDisease Activity Score (modified to include the 28 joint countsaccording to Smolen et al., “Validity and reliability of thetwenty-eight-joint count for the assessment of rheumatoid arthritisactivity.” Arthritis Rheum 38(1):38-43 (1995), which is herebyincorporated by reference in its entirety (DAS28)) were conducted priorto the commencement of the study and at study days 1, 7, 14, 28, 42, 56,84, and at the follow-up visit. The DAS28 consists of a composite scoreof the following variables: tender joint count, swollen joint count,CRP, and Patient's Global Assessment of Disease Activity score (Prevooet al., “Modified Disease Activity Scores that include twenty-eightjoints,” Arthritis & Rheumatism 38(1):44-48 (1995), which is herebyincorporated by reference in its entirety).

The following equation was used to calculate the DAS28 (CRP):

DAS28 (CRP)=0.56√TJC28+0.28√SJC28+0.36·ln(CRP+1)+0.014×(Patient's GlobalAssessment of Disease Activity)+0.96, where:

-   -   TJC28=number of joints tender out of 28    -   SJC28=number of joints swollen out of 28    -   CRP=C-reactive protein    -   Patient's Global Assessment of Disease Activity on a 100 mm        visual analog scale (VAS) recorded by the patient

Interpretation of the DAS28 (CRP) disease activity measure is on a scaleof 0 to 9.4, where: <2.6 is considered remission, ≥2.6 to <3.2 isconsidered low/minimal, ≥3.2 to ≤5.1 is considered moderate, and >5.1 isconsidered high/severe (Anderson et al., “Rheumatoid arthritis diseaseactivity measures: American College of Rheumatology recommendations foruse in clinical practice,” Arthritis Care Res (Hoboken) 64(5):640-7(2012), which is hereby incorporated by reference in its entirety).

Results of this assessment are provided in FIG. 8 and FIG. 9. FIG. 8 isa graph showing the median change in DAS28-CRP from baseline for allpatients as of the assessment date. This data shows a rapid andsustained onset of efficacy, which is markedly different from other p38inhibitors. The increase in DAS28-CRP observed in the follow-up patients(i.e., at day 112) is expected because treatment has stopped andindicates a drug-specific effect. Overall, the maintained decrease inDAS28-CRP observed in all patients through day 84 indicates clinicallyrelevant decrease in disease activity without any evidence oftachyphylaxis. FIG. 9 is a graph showing the percent of patients havinga DAS28-CRP below 2.6 and below 3.2 at each of days 1, 7, 24, 28, 42,56, and 84.

ACR20/50/70. The ACR20 (50/70) response criteria is an endpoint thatindicates the proportion of patients with at least a 20% (50%/70%)improvement in the number of swollen and tender joints (66/68 jointcounts) and at least a 20% (50%/70%) improvement in 3 or more of thefollowing ACR core measures:

To meet the ACR20 criteria, the patient must have at least a 20%improvement in the following ACR Core Set:

-   -   Swollen joint count (66 joint count)    -   Tender joint count (68 joint count)

An improvement of at least 20% in at least 3 of the following 5measures:

-   -   Patient's Global Assessment of Disease Activity (VAS)    -   Patient's Assessment of Arthritis Pain (VAS)    -   Patient's Assessment of Physical Function/Health Assessment        Questionnaire-Disability Index (HAQ-DI)    -   Physician's Global Assessment of Disease Activity (VAS)    -   Acute phase reactant as measured by hsCRP

ACR50=50% improvement in the swollen and tender joint count and at least50% improvement in at least 3 of the 5 measures.

ACR70=70% improvement in the swollen and tender joint count and at least70% improvement in at least 3 of the 5 measures.

Results for this assessment are shown in FIGS. 11-12. The graph of FIG.11 show the median change from baseline in swollen joint count, and thegraph of FIG. 12 shows the median change from baseline in tender jointcount for all patients as of the assessment date. The sustained decreasein swollen joint count and tender joint count over the entirety of thetreatment period (84 days) shows clinically significant decrease indisease activity without any evidence of tachyphylaxis. The increase inswollen and tender joint count (or decrease in change from baseline)observed in the follow-up patients at day 112 was expected becausetreatment had ceased.

The graph of FIG. 14 shows the responder analysis at days 1, 28, 56, 84of treatment and after treatment at day 112. As noted above the ACRvalues represent the proportion of patients in each group meeting ACR20,ACR50, and ACR70 criteria as described above.

RAMRIS and CARLOS Hand MRI Assessments. The Hand-Wrist MRI RAMRIS is avalidated and well-accepted objective imaging measure in RA trials.RAMRIS has sub-scores for synovitis (scored 0 to 3), osteitis (0 to 3),and bone erosions (0 to 10). The CARLOS is a 9-point cartilage lossscale (Perterfy et al., “Monitoring cartilage loss in the hands andwrists in rheumatoid arthritis with magnetic resonance imaging in amulti-center clinical trial: IMPRESS (NCT00425932),” Arthritis Research& Therapy, 15:R44 (2013), which is hereby incorporated by reference inits entirety) that has been used successfully in four randomizedcontrolled clinical trials of RA. The RAMRIS synovitis score changesrapidly with effective RA therapies and changes can be detected even insmall group sizes. In randomized controlled trials, statisticallysignificant decrease in synovitis scores have been observed within 2weeks of onset of effective treatment with approximately 30 patients perarm (Beals et al. “Magnetic resonance imaging of the hand and wrist in arandomized, double-blind, multicenter, placebo-controlled trial ofinfliximab for rheumatoid arthritis: Comparison of dynamic contrastenhanced assessments with semi-quantitative scoring,” PLoS ONE12(12):e0187297 (2017), which is hereby incorporated by reference in itsentirety).

These studies also showed significant inhibition of progression of jointdamage (RAMRIS bone erosion and CARLOS cartilage loss) demonstratedwithin only 12 weeks. The Hand-Wrist MRI scans will be done by the site(or a suitable local facility designated by the site) and will becentrally read. Instructions outlining scan collection parameterssubmission requirements will be provided in a separate site instructionmanual from the MRI central laboratory.

The result of the Hand-Wrist MRI RAMRIS assessments for the currentstudy are provided in FIGS. 15-17 and the tables below. FIG. 15 is agraph showing the percent of patients responsive to ATI-450 treatment asassessed by CARLOS (cartilage loss), erosion, osteitis, synovitis. Thegraph of FIG. 15 is showing percent responsiveness in these endpoints bytreatment and hand. A patient was deemed responsive if a ≥1-pointimprovement in any hand was observed.

FIG. 16 is a graph showing the change in baseline at day 84 for CARLOS,erosion, osteitis, and synovitis in both hands of placebo and ATI-450(50 mg BID) treated patients. The data represents the average of bothhands as assessed by the Hand-Wrist MRI RAMRIS. FIG. 17 is a similargraph showing the change in baseline at day 84 for CARLOS, erosion,osteitis, and synovitis for each hand in placebo and ATI-450 (50 mg BID)patients. Diamonds represent mean values.

Tables 16 and 17 below provide MRI RAMIS assessment scores for CARLOS,erosion, osteitis, and synovitis at baseline and after 12 weeks oftreatment with ATI-450 (50 mg BID) or placebo. The results of thisanalysis collectively demonstrate that ATI 450 treatment prevents theprogression of joint destruction and provides significant improvement insynovitis.

TABLE 16 Hand-Wrist MRI RAMIS Assessment for the Most Severe HandParameter ATI-450 Placebo RAMRIS Synovitis Score Baseline, n 15 2 Mean(SD) 6.1 (4.703) 8.5 (11.314) Week 12, n 15 2 Mean (SD) 5.93 (4.625) 9(12.021) Change from baseline, Mean (SD) −0.17 (2.059) 0.5 (0.707)RAMRIS Osteitis Score Baseline, n 15 2 Mean (SD) 2.93 (4.754) 4.5(6.364) Week 12, n 15 2 Mean (SD) 3.83 (5.063) 6.25 (8.839) Change frombaseline, Mean (SD) 0.9 (2.055) 1.75 (2.475) RAMRIS Erosion ScoreBaseline, n 15 2 Mean (SD) 13.27 (13.625) 17.5 (24.749) Week 12, n 15 2Mean (SD) 13.9 (14.311) 19 (26.87) Change from baseline, Mean (SD) 0.63(1.42) 1.5 (2.121) Carlos Cartilage Loss Baseline, n 15 2 Mean (SD) 7.6(10.677) 18.25 (25.809) Week 12, n 15 2 Mean (SD) 7.63 (10.451) 18.25(25.809) Change from baseline, Mean (SD) 0.03 (0.481) 0 (0)

TABLE 17 Hand-Wrist MRI RAMIS Assessment for the Other Hand ParameterATI-450 Placebo RAMRIS Synovitis Score Baseline, n 15 2 Mean (SD) 4.07(3.914) 7.25 (10.253) Week 12, n 15 2 Mean (SD) 4.4 (4.826) 9.75(13.789) Change from baseline, 0.33 (2.059) 2.5 (3.536) Mean (SD) RAMRISOsteitis Score Baseline, n 15 2 Mean (SD) 2.4 (4.544) 10.25 (14.496)Week 12, n 15 2 Mean (SD) 3.4 (5.562) 10.25 (14.496) Change frombaseline, 1 (2.283) 0 (0) Mean (SD) RAMRIS Erosion Score Baseline, n 152 Mean (SD) 13.3 (13.749) 25.5 (36.062) Week 12, n 15 2 Mean (SD) 13.03(13.2) 25.5 (36.062) Change from baseline, −0.27 (1.178) 0 (0) Mean (SD)Carlos Cartilage Loss Baseline, n 15 2 Mean (SD) 7.45 (16.287) 18.88(26.693) Week 12, n 15 2 Mean (SD) 7.45 (16.287) 18.88 (26.693) Changefrom baseline, 0 (0) 0 (0) Mean (SD)

The Patient's Global Assessment of Disease Activity Visual Analog Scale.The Patient's Global Assessment of Disease Activity VAS is used tomeasure the patient's global assessment of disease activity. The VAS is100 mm in length with “0” (no activity at all, very well) on the leftend of the line and “100” (worst activity imaginable, very poor) on theright end of the line. The participant draws a vertical line through thehorizontal line to indicate their level of disease activity. Study staffmeasure from the left end of the line to the line marked by theparticipant and record this length in mm (0 to 100). The results of thisassessment for treatment (n=13) and placebo (n=2) groups through day 84of the study are show in the graph of FIG. 13A.

Patient's Assessment of Arthritis Pain Visual Analog Scale. ThePatient's Assessment of Arthritis Pain VAS is used to measure thepatient's level of arthritis pain. The VAS is 100 mm in length with “0”(no pain at all, no pain) on the left and “100” (worst pain imaginable,the worst possible pain) on the right end of the line. The participantdraws a vertical line through the horizontal line to indicate how muchpain they are experiencing that day. Study staff measure from the leftend of the line to the line marked by the participant and record thislength in mm (0 to 100). The results of this assessment for treatment(n=13) and placebo (n=2) groups through day 84 of the study are show ingraph of FIG. 13B.

Patient's Assessment of Physical Function/Health AssessmentQuestionnaire-Disability Index. The Patient's Assessment of PhysicalFunction/HAQ-DI is utilized to assess the participant's physicalfunction or disability. The HAQ-DI asks about the degree of difficulty aperson has in accomplishing tasks in 8 functional areas (dressing,arising, eating, walking, hygiene, reaching, gripping, and activities[errands and chores]). Responses in each functional area are scored from“0” indicating no difficulty to “3” indicating inability to perform atask in that area. Study staff should not clarify any of the questionsfor the participant. The results of this assessment for treatment (n=13)and placebo (n=2) groups through day 84 of the study are shown in FIG.13C.

Physician Completed Efficacy Questionnaire. When this assessment iscompleted at study days 1, 28, 56, 84 and at follow-up, it will be oneof the first assessments done at the visit, and must be completed priorto drug dosing. The Physician's Global Assessment of Disease ActivityVAS is a measure completed by the investigator or designee. The VAS is100 mm in length with “0” (no activity at all, no arthritis activity) onthe left end of the line and “100” (worst activity imaginable, extremelyactive arthritis) at the other end of the line. The investigator ordesignee draws a vertical line through the horizontal line to indicatethe patient's disease activity. Study staff measure the line from theleft end to the investigator's (or designee) line and record this lengthin mm (0 to 100 mm). The results of this assessment for treatment (n=13)and placebo (n=2) groups through day 84 of the study are show in thegraph of FIG. 13D.

High Sensitivity C-reactive Protein (hsCRP). Blood samples forevaluation of hsCRP were collected prior to the commencement of thestudy, and at study days 1, 7, 14, 28, 42, 56, 84, and at the time offollow-up (day 112). Samples were shipped to a central laboratory.Specific instructions for collection, processing, storage and shipmentof samples for hsCRP were provided in a separate laboratory manual.

Results of this assessment are provided in FIG. 10. The graph of FIG. 10shows median change in hsCRP from baseline through day 84 for allpatients and through day 112 (i.e., after treatment ceased) for somepatients. The slight increase in hsCRP observed in the follow-uppatients was expected, because treatment had stopped and was indicativeof a drug-specific treatment effect. Overall, the rapid and sustainedreduction in hsCRP from baseline observed in all patients through day 84indicates clinically relevant decrease in disease activity without anyevidence of tachyphylaxis.

Pharmacodynamics. Venous blood samples of approximately 10 mL wascollected for measurement of ex vivo stimulated cytokine levels (e.g.,TNF-α, IL-1β, IL-6, IL-8, IFNγ, IL-17, IL-18, IL-10, IL-1RA, and IL-1α),and phosphoprotein PD parameters at study dates 1, 7, 14, 28, 42, 56,84, and at the follow-up. For the endogenous cytokine levels (e.g.,TNF-α, IL-1β, IL-6, IL-8, IFNγ, IL-17, IL-18, IL-10, IL-1α, and IL-1RA),1 mL of serum will be drawn at each identified time point.

p38 inhibitor compounds as a class have shown a lack of durable efficacyin RA studies. It is thought that dosing with a p38 inhibitor over 12weeks results in pathway reprogramming and inhibitor induced p38independent cytokine production over time. In contrast, administrationof ATI-450 for 12 weeks in RA patients result in sustained inhibition ofproinflammatory cytokines as shown in the graphs of FIGS. 18A and 18B.Blood was analyzed from RA patients on day 1 of dosing and day 84 ofdosing, following ex vivo stimulation with LPS and evaluated for theability of ATI-450 to inhibit TNF-α, IL-1β, IL-6, and IL-8 compared withplacebo. As demonstrated by the last two bars in each cytokine groupshown in FIGS. 18A and 18B, ATI-450 inhibition of each cytokine after 84days of dosing was very similar to ATI-450 inhibition of each cytokineof each cytokine after 1 day of dosing. This data shows that inhibitordependent pathway reprogramming and tachyphylaxis is not observed withthe MK2 inhibitor ATI-450, in contrast to what has been hypothesizedwith p38 inhibitors.

Endogenous cytokines were evaluated in plasma from the RA patients overthe course of dosing ATI-450 for 12 weeks. The data for TNF-α and IL-1RAare provided in FIGS. 19A and 19B, respectively. These data areexpressed as median % change relative to pre-dosing for bothdrug-treated and placebo-treated patients. Samples were evaluated attrough drug levels through day 84 and the last point is near C_(max)following the final dose on day 84. The horizontal line (

) in FIG. 19A depicts TNF-α levels in blood from normal volunteersubjects that were evaluated in the same assay. TNF-α levels are rapidlyreduced by 4 weeks of dosing (trough) and this reduction was maintainedthroughout the 12 weeks of dosing. The horizontal line (

) in FIG. 19B represents IL-1RA levels in healthy subjects. In contrastthe anti-inflammatory cytokine IL-1RA was elevated in RA patients and isnot impacted by ATI-450 across the 12 weeks. This data indicates thatATI-450 cytokine inhibition is skewed toward proinflammatory cytokinesrelative to anti-inflammatory cytokines.

FIGS. 20A-20D provide a broader look at the impact of ATI-450 onendogenous cytokine levels in plasma from the RA patients over thecourse of dosing ATI-450 for 12 weeks. Plasma levels of threeproinflammatory cytokines (TNF-α (FIG. 20A), IL-6 (FIG. 20C), IL-8 (FIG.20B)), from a 13-plex tested are shown in this Figure, along with thechemokine MIP1β (FIG. 20D). The data are expressed as median % changerelative to pre-dosing for drug treated patients. Samples were evaluatedat trough drug levels after 4, 8 and 12 weeks of dosing and 4 hoursafter the final dose on day 84. The horizontal line (

) describes cytokine/chemokine level in plasma from normal healthyvolunteer subjects. TNF-α levels are significantly reduced after 4 weeksof dosing and this reduction was maintained throughout the 12 weeks andapproached the level of cytokine present in healthy volunteers.Similarly, IL-6, IL-8, and MIP113 levels are reduced at week 4 and thisreduction was maintained through the 12 weeks of the study. Thisanalysis further demonstrates the durable anti-inflammatory activity ofATI-450 dosed at 50 mg BID in RA patients over 12 weeks.

Safety. Overall, the study drug was very well tolerated, with no seriousor severe adverse events reported. Only one subject withdrew forinvestigation of palpitations and elevated CPK. Table 18 belowsummarizes the reported events.

TABLE 18 Adverse Events Reported by Study Subject Preferred Term ATI-45050 mg BID (n = 16) Placebo (n = 3) Mild Moderate Mild Moderate (n %) (n%) (n %) (n %) Blood cholesterol 1 (6.25) 0 increased Blood creatine 0 1(6.25) phosphokinase increased Constipation 1 (6.25) 0 Dental caries 1(33.33) 0 Ear infection 1 (6.25) 0 Abnormal ECG 1 (6.25) 0 Essential 0 1(6.25) hypertension Hyperliidaemia 0 1 (6.25) Hypokalaemia 0 1 (6.25)Ligament sprain 1 (6.25) 0 Low density 1 (6.25) 0 lipoprotein increasedMouth ulceration 1 (6.25) 0 Muscle strain 0 1 (33.33) Palpitations 1(6.25) 0 Rash 1 (6.25) 0 erythematous Sinusitis 0 1 (6.25) Skin abrasion1 (6.25) 0 Urinary tract 0 2 (12.5) infection Ventricular 1 (6.25) 0extrasystoles White blood 1 (6.25) 0 cell count increased

Summary of the Results:

ATI-450 was generally well tolerated. One subject in the treatment armwithdrew due to palpitations, which were unrelated to the studymedication, and an elevated CPK, which was determined by the siteinvestigator to be drug-related. One subject in the placebo arm withdrewas a result of prohibited medication needed to treat muscle strain. Noserious adverse events were reported and all adverse events were mild tomoderate. The most common adverse events (reported by at least 2subjects) were urinary tract infection and elevated lipids.

In this trial, ATI-450 demonstrated durable clinical activity, asdefined by a marked and sustained reduction in DAS28-CRP and evaluationof ACR20/50/70 responses over 12 weeks. The mean change from baseline inDAS28-CRP at week 12 was a 2.0 reduction in the treatment arm and a 0.35increase in the placebo arm. The proportion of subjects with a DAS28-CRPscore at week 12 of ≤ to 3.2 (low disease activity or remission) was 40%and 0% in the 15 subjects in the treatment arm and 2 subjects in theplacebo arm, respectively, of which the proportion of subjects with aDAS28-CRP score of <2.6 (remission) was 20% and 0% in the treatment andplacebo arms, respectively.

ACR20/50/70 was observed at week 12 in 60%, 33% and 20%, respectively,of the 15 subjects in the treatment arm, and in 0% of two subjects inthe placebo arm. The median reduction from baseline in hsCRP was >40%throughout the 12 weeks of the trial in the treatment arm. An interimanalysis (11 treatments, two placebo) of ex vivo stimulated cytokinesfrom blood samples taken from the treatment arm showed a marked anddurable inhibition of TNF-α, IL-10, IL-6, and IL-8 over the 12 weekdosing period. Similarly, analysis of endogenous cytokines alsodemonstrated a marked and sustained inhibition of median concentrationsof TNF-α, IL-6, IL-8, and MIP1β in the treatment arm over the 12-weekperiod.

ATI-450 was generally well tolerated. No serious adverse events werereported and all adverse events were mild to moderate. The most commonadverse events (each reported in 2 subjects) were urinary tractinfection (UTI), elevated lipids and ventricular extrasystoles, all ofwhich were determined to be unrelated to treatment except for one UTI.Two subjects withdrew from the trial, one in the treatment arm and onein the placebo arm.

Example 5 Phase 2B, Randomized, Double-Blind, Parallel Group,Placebo-Controlled Study to Investigate Efficacy, Safety, Tolerability,Pharmacokinetics, and Pharmacodynamics of Multiple Doses of ATI-450 PlusMTX Versus MTX Alone in Patients with Moderate to Severe RA

This is a Phase 2b, randomized, double-blind, parallel group,placebo-controlled study to investigate the efficacy, safety,tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) ofmultiple doses of ATI-450 plus MTX versus MTX alone in patients withmoderate to severe RA who have had an inadequate response tomethotrexate alone.

Subjects whose eligibility is confirmed at baseline will be randomizedin a 1:1:1:1 ratio to receive either ATI-450 tablets (20 mg, 50 mg, and80 mg twice daily [BID]) plus MTX, or matching placebo tablets BID plusMTX. The randomization of patients to treatment groups will bestratified by two separate factors. The first stratification factor willbe patients who test positive for rheumatoid factor (RF) and/oranti-cyclic citrullinated peptide (CCP) at screening versus thosepatients who do not test positive for both of these antibodies. Thesecond stratification factor will be those patients who have experiencewith either a biologic RA treatment or a JAK RA treatment versus thosewith no such experience. Enrollment will be limited such that no morethan 25% of the study population will have tested negative for either RFor anti-CPP or both. Likewise, enrollment will be limited such that nomore than 25% of the population has prior experience with either abiologic RA therapy or a JAK RA therapy. Study medications will beadministered orally for 12 weeks. Patients will be required to remain ona stable dose of MTX (15 mg to 25 mg/week for 4 weeks prior to screen or10 mg/week for 4 weeks prior to screening for patients with documentedintolerance) for the duration of the study.

Patients will attend clinic visits on Days 1, 8, 15, 29, 43, 57, and 85(±1 day) for safety, efficacy, trough PK, and PD assessments. Themorning dose of study medication will be administered in the clinic oneach study visit day.

On Day 85 (Week 12), patients will complete the end of studyassessments. A safety follow-up visit will be conducted 30 days (+7)after the last dose of study medication.

Inclusion criteria for subjects include 1) a diagnosis of adult-onsetrheumatoid arthritis (RA) as defined by the 2010 American College ofRheumatology/European League Against Rheumatism classification criteria;2) Disease Activity Score using 28 join count-C-reactive protein (CRP)(DAS28-CRP) greater than 3.2 defined as moderate or high diseaseactivity; 3) have moderate to severe RA defined by a minimum diseaseactivity criteria (6 or more swollen joints at screening and baselinevisits; 6 or more tender joints at screening and baseline visits; and ahigh-sensitivity C-reactive protein (hsCRP) level greater than the upperlimit of normal (ULN) or positive for both RF and/or anti-cycliccitrullinated peptide (CCP) at screening visit); 4) a minimum of 12weeks on MTX with a stable MTX dose (of 15 mg to 25 mg/week or 10mg/week for patients with documented intolerance) for at least 4 weeksprior to screening; 5) male or non-pregnant, non-nursing female aged18-75 years old (inclusive); and 6) screening laboratory evaluationsfalling within the normal range of the central laboratory's referenceranges.

Efficacy Analyses

All efficacy summaries will be conducted on both the Intent-to-treat(ITT) and Per-protocol (PP) populations.

The primary endpoint assessed in this study is the proportion ofsubjects achieving American College of Rheumatology (ACR) 20 at 12weeks.

Secondary endpoints to be assess in this study include: 1) proportion ofsubjects with ACR 50/70 at week 12; 2) proportion of subjects with ACR20/50/70 over time; 3) Mean change from baseline in Disease ActivityScore using 28 joint count-C-reactive protein (CRP) (DAS28-CRP) overtime; 4) Proportion of patients achieving DAS 28 remission (score <2.6)over time and at week 12; 5) Mean change from baseline in ClinicalDisease Activity Index (CDAI) over time; 6) Median percent change frombaseline in high sensitivity C-reactive protein (hsCRP) levels overtime; 7) Health Assessment Questionnaire Disability Index (HAQ-DI) overtime; 8) ATI-450 concentrations at clinic visits (trough and 2-hour postdose sampling for all patients; and additional serial sampling fornoncompartmental PK analysis for patients in PK sub-study); 9) Meanchange from baseline in endogenous cytokine levels (e.g., TNF-α, IL-1β,IL-6, IL-9, IFNγ, IL-17, IL-18, IL-10, IL-1α, and IL-1RA).

ACR 20/50/70 responder rates as well as the proportion of patients withDAS28-CRP <2.6 will be analyzed using a logistic regression. Patientswith missing ACR values or missing DAS28-CRP values will be considerednon-responders. Patients with negative intercurrent events (such asrequiring rescue) will also be considered non-responders for ACR20/50/70 and DAS28-CRP <2.6.

The DAS28-CRP, CDAI, 66/68 swollen/tender joint count, Patient's GlobalAssessment of Disease Activity, Patient's Assessment of Arthritis Pain,and Physicians Global Assessment of Disease Activity, as well as theircorresponding changes from baseline will be analyzed over time (for allscheduled time points) using mixed model repeated measures. Model basedpoint estimates and 95% confidence intervals will be provided fortreatment effects. Point estimates, 95% confidence intervals andp-values will be provided for treatment differences versus placebo.

The hsCRP levels, change from baseline in hsCRP and percent change frombaseline in hsCRP will be summarized over time using continuousstatistical summary measures. Determination of sustained treatmenteffect for hsCRP will be based upon the median percent change frombaseline in hsCRP over time in the PP population. Exploratory doseresponse modeling will be conducted for ACR 20/50/70 responder rates,the change from baseline in DAS28-CRP, 66/68 swollen/tender joint count,Patient's Global Assessment of Disease Activity, Patient's Assessment ofArthritis Pain, the Physicians Global Assessment of Disease Activity, aswell as percent change from baseline in hsCRP, separately.

Safety of treatment will be based on 1) number and frequency of adverseevents (AEs), 2) vital signs, 3) ECG, 4) serum chemistry, 5) hematology,6) urinalysis, and 7) coagulation.

Plasma concentrations of ATI-450 and its primary circulating metabolite(CDD-2164) will be measured on each assessment day.

Example 6 Phase 2B, Randomized, Double-Blind, Parallel Group,Placebo-Controlled Study to Investigate Efficacy, Safety, Tolerability,Pharmacokinetics, and Pharmacodynamics of Multiple Doses of ATI-450 vsPlacebo in Patients with Moderate to Severe Active Psoriatic Arthritis

This is a Phase 2b, randomized, double-blind, parallel group,placebo-controlled study to investigate the safety, tolerability, PK,and PD of multiple doses of ATI-450 versus placebo in patients withmoderate to severe active psoriatic arthritis (PSoA) who have had aninadequate response to MTX alone.

Subjects meeting eligibility criteria will receive 50 mg ATI-450 twicedaily or a matching placebo tablet twice daily. Treatment will continuefor 12 weeks while remaining on a stable dose of MTX (15 mg to 25 mg perweek) prior to and for the duration of the study. PK and PD assessmentsoccurred on days 1, 8, 15, 29, 43, 57 and 85 days. Subject groups arestratified based on prior use of DMARDs (Disease ModifyingAnti-Rheumatic Drug).

Inclusion criteria for subjects include 1) a clinical diagnosis of PSoAwith symptom onset at least 6 months prior to the screening visit andfulfillment of the Classification Criteria for PsA (CASPAR criteria); 2)An active disease at baseline defined as at least 3 tender joints and atleast 3 swollen joints at screening and baseline visits; 3) Presence ofeither at screening: (a) at least one erosion on X-ray as determined bycentral imaging review; or (b) hsCRP level higher than thelaboratory-defined ULN; 4) A diagnosis of active plaque psoriasis ordocumented history of plaque psoriasis; 5) An inadequate response (lackof efficacy after a minimum 12 week duration of therapy) to previous orcurrent treatment with at least 1 non-biologic DMARD at a maximallytolerated dose (MTX, sulfasalazine, leflunomide, cyclosporine,apremilast, bucillamine, or iguratimod), or an intolerance orcontraindicated for DMARDs; and 6) Subjects being treated concurrentlywith non-biologic DMARDs at study entry must be on less than or equal to2 non-biologic DMARDs (except the combination of MTX and leflunomide).The following DMARDs are permitted: MTX, sulfasalazine, leflunomide,apremilast, hydroxychloroquine, bucillamine, and iguratimod. Treatmentsmust have been ongoing for at least 12 weeks at a stable dose for atleast 4 weeks prior to the baseline visit. Finally, screening laboratoryevaluations (hematology, chemistry, coagulation, and urinalysis) mustfall within the normal range of the central laboratory's referenceranges.

Exclusion Criteria for subjects include: 1) Current treatment with morethan two non-biologic DMARDs; the use of DMARDs other than methotrexate,sulfasalazine, leflunomide, apremilast, hydroxychloroquine, bucillamine,and iguratimod; or the use of methotrexate in combination withleflunomide. 2) A history of fibromyalgia, any arthritis with onsetprior to age 17 years, or current diagnosis of inflammatory jointdisease other than PsA (including, but not limited to rheumatoidarthritis, gout, overlap connective tissue diseases, scleroderma,polymyositis, dermatomyositis, systemic lupus erythematosus). A priorhistory of reactive arthritis or axial spondyloarthritis includingankylosing spondylitis and non-radiographic axial spondyloarthritis ispermitted if documentation of change in diagnosis to PsA or additionaldiagnosis of PsA is made. A prior history of fibromyalgia is permittedif documentation of change in diagnosis to PsA or documentation that thediagnosis of fibromyalgia was made incorrectly. 3) a current acute orchronic immunoinflammatory disease other than PSoA which may impact thecourse or assessment of PSoA. 4) An uncontrolled non-immunoinflammatorydisease that may place the patient at increased risk during the study orimpact the interpretation of results. 5) A history or evidence of activeor latent tuberculosis. 6) A history of alcoholism/current alcoholic,alcoholic liver disease, or other chronic liver disease; activetreatment of an infection with antibiotics. 7) Positive for HIV orhepatitis B or C. 8) a white blood cell count of less than 3.0×10³cells/mm³; an absolute neutrophil count of less than 1.5×10³ cells/mm³;a lymphocyte count of less than 0.5×10³ cells/mm³; a platelet count ofless than 100×10³ cells/mm³; a hemoglobin level less than 10 g/dL; anaspartate aminotransferase (AST) or alanine aminotransferase (ALT) levelequal to or greater than 1.5× ULN; a total bilirubin level equal to orgreater than 2× ULN unless patient has been diagnosed with Gilberts'disease and this is clearly documented; an estimated glomerularfiltration rate (eGFR) of less than 40 mL/min/1.73 m² based onModification of Diet and Renal Disease formula. 9) A blood pressurelevel (in supine position after at least 5 minutes rest) less than 90mmHg or greater than 140 mmHg for systolic blood pressure or less than40 mmHg or greater than 90 mmHg for diastolic blood pressure. 10) Hastaken rituximab or other B cell inhibitor within 6 months; has taken abiologic DMARD within 8 weeks; has taken enteracept or anakinra within 4weeks; has taken a conventional DMARD (leflunomide, cyclosporine,azathioprine) or JAK inhibitors within one month. 11) Has had anintra-articular corticosteroid injection within 30 days; has taken aninvestigational small molecule within 30 days; are currently receivingcorticosteroids at doses greater than 10 mg per day of prednisone (orequivalent) or have been receiving an unstable dosing regimen ofcorticosteroids within 2 weeks of the screening visit; have startedtreatment with non-steroidal anti-inflammatory drugs (NSAIDs) or havebeen receiving an unstable dosing regimen of NSAIDs within 2 weeks ofthe screening visit. 12) a history of stroke; a significant cardiacdisease that would affect interpretation of study data or the safety ofthe patient's participation in the study, per judgment of theinvestigator, including recent myocardial infarction or unstable angina,or heart failure with New York Heart Association Class III or IVsymptoms; evidence of atrial fibrillation, atrial flutter, completeright or left bundle branch block, Wolff-Parkinson-White Syndrome, orother significant rhythm disturbance; evidence of acute ischemia;screening or pre-dosing baseline mean QTcF greater than 450 msec; apersonal or family history of long QT syndrome or sudden death. 13) anyjoint procedure within 90 days of screening.

Efficacy Analyses:

The primary objective of this study is to assess the efficacy ofmultiple doses of ATI-450 in patients with moderate to severe PSoA. Thisobjective will be assessed by determining the proportion of subjectsachieving ACR (American College of Rheumatology) 20 at week 12 in theITT population. Response defined as at least 20% reduction (improvement)compared with baseline in tender joint count (TJC), swollen joint count(SJC), and at least 3 of the 5 remaining ACR core set measures:patient's assessment of pain, patient's global assessment of diseaseactivity (PtGA); physician's global assessment of disease activity(PhGA), Health Assessment Questionnaire-Disability Index (HAQ-DI), andhigh sensitivity C-reactive protein (hsCRP).

The secondary objective of this study is to assess the pharmacodynamics(PD) of ATI-450 in patients with moderate to severe PSoA. This objectivewill be assessed by determining 1) proportion of patients with ACR 50/70at week 12; 2) proportion of patients with ACR 20/50/70 over time; 3)proportion of patients achieving a static Investigator Global Assessment(sIGA) of Psoriasis of 0 or 1 and at least a 2-point improvement frombaseline; 4) a psoriasis area severity index (PAST) 75 response (forparticipants with at least a 3% BSA psoriasis as baseline); 5)proportion of participants achieving minimal disease activity (MDA),determined based on subjects fulfilling 5 of 7 outcome measures: TJC≤1;SJC≤1; PASI≤1 or BSA-Ps≤3%; Patient's Assessment of Pain NRS≤1.5;PtGA-Disease Activity NRS≤2.0; HAQ-DI score≤0.5; and tender enthesealpoints≤1; 5) a change from baseline in HAQ-DI over 12 weeks; 6) a changefrom baseline in short-form (SF)-36 Physical Component Summary (PCS); 7)a change from baseline FACIT-Fatigue Questionnaire; 8) a change frombaseline in Self-Assessment of Psoriasis Symptoms (SAPS) Questionnaire;and 9) safety (adverse effects, ECG, laboratory values). Anothersecondary objective of this study is to assess the pharmacokinetics (PK)of ATI-450 in patients with moderate to severe PSoA who are receivingconcomitant MTX. This objective will be assessed by determining theATI-450 concentrations at clinic visits (trough PK analysis).

An exploratory objective of this study is to assess the PD of ATI-450plus MTX in patients with moderate to severe PSoA. This objective willbe assessed by determining Mean change from baseline in endogenouscytokine levels (e.g., tumor necrosis factor-α [TNF-α], interleukin[IL]-1β, IL-6, IL-8, IFNγ, IL 17, IL-18, IL-10, IL-1α and IL-1RA)

All efficacy summaries will be conducted on both the ITT and PPpopulations.

The primary efficacy analysis will be the treatment comparison of thepercentage of patients achieving ACR20 at week 12 in the ITT population.This analysis will be conducted within the context of a logisticregression model. Treatment group will enter the model as a classvariable and baseline severity will be included as a covariate. Thehypothesis test and corresponding p-value will be a one-sided test withan alpha level of 0.05. Point estimates and 90% confidence intervals forthe odds ratio will be provided in addition to the p-values.

Treatment comparisons for all secondary endpoints will be conductedusing a logistic regression like that of the primary analysis. This willbe done on both the ITT and PP populations.

Treatment comparisons for all continuous secondary endpoints will beconducted using a Mixed Model Repeated Measures (MMRM). This model willinclude factors for treatment group, time, and time by treatmentinteraction as well as a baseline covariate, where appropriate. Subjectidentifier will be included in the model in a manner that allowsobservations within a given subject over time to be treated as repeatedmeasures. All treatment comparisons will be based on a one-sidedhypothesis test with a 0.05 alpha level. Point estimates and 90%confidence intervals for the difference in least square means will beprovided in addition to the p-values. These analyses will be done forboth the ITT and PP populations.

Safety of treatment will be based on 1) incidence and severity of AEs,vital signs, ECG measurements, clinical laboratory values (hematology,serum chemistry, coagulation, urinalysis).

Example 7 A Phase 2a, Randomized, Double-Blind, Parallel Group,Placebo-Controlled Dose Ranging Study to Investigate the Efficacy,Safety, Tolerability, Pharmacokinetics, and Pharmacodynamics of ATI-450vs Placebo in Patients with Moderate to Severe Hidradenitis Suppurativa(HS)

This is a Phase 2b, randomized, double-blind, parallel group,placebo-controlled study to investigate the efficacy, safety,tolerability, PK, and PD of ATI-450 50mg BID versus placebo in patientswith moderate to severe HS who have had an inadequate response orintolerance to an adequate trial of oral antibiotics. Subjects meetingeligibility criteria will receive 50 mg or 80 mg ATI-450 (oral tablet)twice daily or a matching placebo tablet twice daily. PK and PDassessments occurred on days 1, 8, 15, 29, 43, 57 and 85 days.

Inclusion criteria include: 1) subject diagnosed with moderate to severeHidradenitis Suppurativa (HS) for at least one year prior to baselinevisit; 2) subject exhibits HS lesions (Hurley stage II or III) presentin at least two distinct anatomical areas; 3) draining fistula count of<=20 at Baseline visit; 4) total abscesses and nodule count (AN count)of >=5 at Baseline visit; 5) subject agrees to use a daily antisepticwash on their HS lesions; 6) subject must have a history of inadequateresponse or intolerance to an adequate trial of oral antibiotics fortreatment of HS.

Exclusion criteria include a history of active skin disease other thanHS that could interfere with the assessment of HS; an uncontrollednon-immunoinflammatory disease that may place the patient at increasedrisk during the study or impact the interpretation of results; a historyor evidence of active or latent tuberculosis; a history ofalcoholism/current alcoholic, alcoholic liver disease, or other chronicliver disease; active treatment of an infection with antibiotics;positive for HIV or hepatitis B or C; a white blood cell count of lessthan 3.0×103 cells/mm3; an absolute neutrophil count of less than1.5×103 cells/mm3; a lymphocyte count of less than 0.5×103 cells/mm3; aplatelet count of less than 100×103 cells/mm3; a hemoglobin level lessthan 10 g/dL; an aspartate aminotransferase (AST) or alanineaminotransferase (ALT) level equal to or greater than 1.5× ULN; 12) atotal bilirubin level equal to or greater than 2× ULN unless patient hasbeen diagnosed with Gilberts' disease and this is clearly documented; anestimated glomerular filtration rate (eGFR) of less than 40 mL/min/1.73m2 based on Modification of Diet and Renal Disease formula; a bloodpressure level (in supine position after at least 5 minutes rest) lessthan 90 mmHg or greater than 140 mmHg for systolic blood pressure orless than 40 mmHg or greater than 90 mmHg for diastolic blood pressure;has taken rituximab or other B cell inhibitor within 6 months; has takena biologic DMARD within 8 weeks; has taken enteracept or anakinra within4 weeks; has taken a conventional DMARD (leflunomide, cyclosporine,azathioprine) or JAK inhibitors within one month; an intra-articularcorticosteroid injection within 30 days; has taken an investigationalsmall molecule within 30 days; is currently receiving corticosteroids atdoses greater than 10 mg per day of prednisone (or equivalent) or havebeen receiving an unstable dosing regimen of corticosteroids within 2weeks of the screening visit; have started treatment with non-steroidalanti-inflammatory drugs (NSAIDs) or have been receiving an unstabledosing regimen of NSAIDs within 2 weeks of the screening visit; ahistory of stroke; a significant cardiac disease that would affectinterpretation of study data or the safety of the patient'sparticipation in the study, per judgment of the investigator, includingrecent myocardial infarction or unstable angina, or heart failure withNew York Heart Association Class III or IV symptoms; evidence of atrialfibrillation, atrial flutter, complete right or left bundle branchblock, Wolff-Parkinson-White Syndrome, or other significant rhythmdisturbance; evidence of acute ischemia; screening or pre-dosingbaseline mean QTcF greater than 450 msec; a personal or family historyof long QT syndrome or sudden death; and any joint procedure within 90days of screening.

Efficacy Analysis

The primary objective of this study is assess the efficacy of multipledoses of ATI-450 in patients with moderate to severe HS. This objectivewill be assessed by determining percentage of patients achievingHidradenitis Suppurativa Clinical Response (HiSCR) at week-12 (HiSCR isdefined as at least a 50% reduction from Baseline in the total abscessand inflammatory nodule (AN) count, with no increase in abscess ordraining fistula counts).

A secondary objective of this study is to assess the efficacy and safetyof ATI-450 in patients with moderate to severe HS. This objective willbe assessed by determining 1) percentage of patients achieving at least30% reduction from Baseline in Numerical Rating Scale (NRS30) inPatient's Global Assessment of Skin Pain (PGA Skin Pain) at week 12among patients with baseline NRS30≥3 (NRS30 is evaluated based on worstskin pain in a 24-hour recall period (maximal daily pain)); 2)percentage of patients achieving at least 30% reduction from baseline inNRS30 in PGA Skin Pain at week 12 among patients with baseline NRS30≥3;3) percentage of patients who experience at least 25% increase in ANcounts with a minimum increase of 2 relative to baseline over the12-week treatment period; 4) change from baseline in Dermatology LifeQuality Index (DLQI) over 12 weeks (the DLQI is a 10-item validatedquestionnaire used to assess the impact of HS disease symptoms andtreatment on quality of life (QoL). It consists of 10 questionsevaluating impact of skin diseases on different aspects of aparticipant's QoL over the prior week, including symptoms and feelings,daily activities, leisure, work or school, personal relationships, andthe side effects of treatment); 5) change from baseline in HS-relatedswelling assessed based on the Hidradenitis Suppurativa SymptomAssessment (HSSA) over 12 weeks (HSSA is a 9-item patient reportedoutcome (PRO) questionnaire developed to assess the symptoms of HS on a0 to 11-point NRS, where 0 represents no symptoms and 10 representsextreme symptom experience); 6) change from baseline in HS-related odorassessed based on the HSSA over 12-weeks; 7) change from baseline inHS-related worst drainage assessed based on the HSSA over 12-weeks; and8) safety (AEs, SAEs, laboratory values, ECG). Another secondaryobjective of this study is to assess the pharmacokinetics (PK) andpharmacodynamics (PD) of ATI-450 in patients with HS. This objectivewill be assessed by determining ATI-450 concentrations at clinic visits(trough PK analysis).

Pharmacodynamics of ATI-450 in patients with moderate to severe HS willalso be assessed by determining the mean change from baseline inendogenous cytokine levels (e.g., TNF-α IL-1β, IL-6, IL-8, IFNγ, IL 17,IL-18, IL-10, IL-1α, and IL-1RA).

All efficacy summaries will be conducted on both the ITT and PPpopulations.

The primary efficacy analysis will be the treatment comparison of thepercentage of patients achieving HiSCR at week 12 in the ITT population.This analysis will be conducted within the context of a logisticregression model. Treatment group will enter the model as a classvariable and baseline severity will be included as a covariate. Thehypothesis test and corresponding p-value will be a one-sided test withan alpha level of 0.05. Point estimates and 90% confidence intervals forthe odds ratio will be provided in addition to the p-values.

Treatment comparisons for all secondary responder endpoints (percentageof patients achieving at least 30% reduction from Baseline in NRS30 inPGA Skin Pain and percentage of patients who experience at least 25%increase in AN count with a minimum increase of 2 relative to baselineover the 12-week treatment period) will be conducted using a logisticregression like that of the primary analysis. This will be done on boththe ITT and PP populations.

Treatment comparisons for all continuous secondary endpoints will beconducted using a Mixed Model Repeated Measures (MMRM). This model willinclude factors for treatment group, time, and time by treatmentinteraction as well as a baseline covariate, where appropriate. Subjectidentifier will be included in the model in a manner that allowsobservations within a given subject over time to be treated as repeatedmeasures. All treatment comparisons will be based on a one-sidedhypothesis test with a 0.05 alpha level. Point estimates and 90%confidence intervals for the difference in least square means will beprovided in addition to the p-values. These analyses will be done forboth the ITT and PP populations.

Example 8 A Phase 2a, Open-Label, Single-Arm Study to Investigate theSafety and Efficacy of ATI-450 for the Maintenance of Remission inPatients with Cryopyrin-Associated Periodic Syndrome (CAPS) PreviouslyManaged with Anti-IL-1 Therapy

CAPS is a rare hereditary autoinflammatory disease caused by again-of-function mutation of the NLRP3 gene coding for cryopyrin, whichis a component of the NLRP3 inflammasome. Dysregulation of the NLRP3inflammasome results in an overproduction of interleukin-1 (IL-1), andconsequently the inflammatory symptoms seen in CAPS. Current anti-IL-1therapies have been shown to induce rapid and sustained diseaseremission in CAPS patients and are generally well tolerated.

ATI-450 has been shown to cause a marked inhibition of IL-1 in bothpre-clinical and clinical studies. Current anti-IL-1 therapies fortreatment of CAPS are biologics that are administered eitherintravenously or subcutaneously. ATI-450, an orally administered smallmolecule, has the potential to have a similar safety and efficacyprofile compared to currently available therapies with a differentiatedroute of administration that may be preferred by some CAPS patients.

This study is being conducted to determine the safety, tolerability,pharmacokinetics (PK), pharmacodynamics (PD), and preliminary efficacyof ATI-450 in patients with CAPS.

The primary objective of this study is to assess the safety andtolerability of ATI-450 to maintain remission in patients with CAPSpreviously managed with anti-IL-1 therapy. This objective will beassessed by investigating the number and percent of adverse events (AEs)and serious adverse events (SAEs); mean change from baseline inlaboratory values, vital signs, and electrocardiograms (ECGs).

The secondary objective of this study is to assess the efficacy ofATI-450 to maintain remission in patients previously managed withanti-IL-1 therapy. To assess this objective, several endpoints will beassessed. These endpoints include (i) determining the proportion ofparticipants in disease remission over time. Disease remission isdefined as having a Physician Global Assessment (PGA) score of absent orminimal and a high sensitivity C-reactive protein (hsCRP) and serumamyloid A (SAA) value within the normal range (≤10 mg/L) or within 30percent of the baseline value. (ii) Determining the proportion ofparticipants in clinical remission over time. Clinical remission isdefined as having a PGA score of absent or minimal. (iii) Determiningthe time to relapse; where relapse is defined as a two-point worseningon the PGA scale. (iv) Determining the proportion of participants whoexperience re-emergence of disease symptoms after discontinuation ofATI-450. Re-emergence is defined as a daily Key Symptom Score (KSS) ≥3points higher than baseline for at least 2 consecutive days. KSS isderived from the patient-administered daily health assessment form(DHAF). (v) Determining the proportion of participants with a mean KSSno more than 2 points higher than baseline for at least 6 out of 8 weeksduring the treatment period. (vi) Determine change from baseline in PGA.(vii) Determine change from baseline in KSS. (viii) Determine changefrom baseline in hsCRP and SAA.

In addition to the above, the study will also investigate the followingtwo exploratory objectives: (i) assessing the pharmacodynamics (PD) ofATI-450 in patients with CAPS, and (ii) assessing the Pharmacokinetics(PK) of ATI-450 in patients with CAPS. These objectives will be assessedby determining the change from baseline in serum cytokines IL-1β, IL-1α,IL-6, IL-18, and TNF-α and the ATI-450 concentrations at trough.

This is a Phase 2a, open-label, single-arm study to investigate thesafety, tolerability, efficacy, PK, and PD of ATI 450 to maintainremission in patients with CAPS previously managed with anti-IL-1therapy. Up to 10 patients are planned to be enrolled in the study. Thestudy will consist of an up to 8-week screening period, a 12-weektreatment period, and a 4-week safety follow-up period. The totalduration of the study for patients remaining in the study until theirfinal safety follow up assessment will be up to 24 weeks.

The investigator will obtain signed informed consent from the patientbefore any study procedures are performed. For further details regardingthe informed consent process, see Section 9.3. During the screeningvisit each patient will be required to have all assessments performed asoutlined in the Schedule of Assessments (Table 13).

Patients eligible for the study include those with (1) Diagnosis ofFamilial Cold Autoinflammatory Syndrome, Muckle-Wells Syndrome, orNeonatal Onset Multisystem Inflammatory Disease. Prior agreement betweenthe Investigator and Aclaris for study eligibility is required forpatients who do not have a molecular diagnosis of NLRP3 mutationsavailable (either testing not performed, or testing performed, butnegative) upon study entry. For those patients who have not beenmolecularly tested for NLRP3 mutations, molecular testing should beperformed during the study; (2) Patients with a PGA score of “minimal”or less and who are considered to have achieved that response as aresult of successful anti-IL-1 therapy; and (3) Continuous Treatmentwith anti-IL-1 therapy for at least 6 months.

Patients whose eligibility is confirmed at baseline will begin dosingATI-450 tablets (50 mg BID). Such patients will be in protocol-definedremission due to prior treatment with an anti-IL-1 biologic. Dosing willbegin on the day that the next dose of their anti-IL-1 therapy isscheduled at which time anti-IL-1 therapy will be discontinued.

The first dose of ATI-450 (50 mg BID) will be taken at the followingtimes (based on anti-IL-1 therapy):

Anakinra: 24 hours (+/−1 hour) after last administration or atapproximate time of regular anakinra dose.

Canakinumab: 8 weeks (+/−1 week) after last administration.

Rilonacept: 1 week (+/−1 day) after last administration.

ATI-450 will be administered orally for 12-weeks. Patients will attendclinic visits on Days 14, 28, 56, and 84 (+/−1 day) for safety,efficacy, PK and PD assessments. Patients will begin recording diseasesymptoms in their daily diary card 7 days prior to beginningadministration of ATI-450, or immediately starting at the screeningvisit if the Day 1 visit is scheduled to occur less than 7 days afterthe screening visit. Patients will continue to complete their diaryuntil completion of the safety follow up Day 7 visit.

At the end of 12-weeks, Day 84 (+/−1 day), patients will stop ATI-450and conduct end of study assessments. Patients will complete safetyfollow-up visits on site 7 Days (+4 days) after the last dose of ATI-450and via a phone call 30 Days (+/−3 days) after the last dose.

The study is designed to assess the safety, tolerability, efficacy, PK,and PD of ATI-450 in patients with CAPS.

Many patients with CAPS achieve complete or near complete diseaseremission with currently available anti-IL-1 therapies. Therefore, thestudy is designed to minimize patient's time off anti-IL-1 therapy dueto potentially harmful long-term complications that can result fromprolonged relapse of disease symptoms.

The study is open-label and there is no washout period for anti-IL-1therapy; patients enrolled in the trial will begin treatment with studymedication (Day 1) on the day of their next scheduled dose of anti-IL-1therapy. At the end of the 12-week treatment period, ATI-450administration will be discontinued, and patients will delayadministration of anti-IL-1 therapy until 7 days (+4 days) after thelast dose of ATI-450.

The 12-week treatment period was selected to allow sufficient time toexplore the efficacy of ATI-450 in maintaining disease remission, and toadequately rule out the potential for prolonged disease remission causedby the last dose of anti-IL-1 therapy. Due to its long t_(1/2), this isparticularly relevant in instances when canakinumab is used.

The start of the study will be the date on which the first patientprovides informed consent, and the end of the study will be the date ofthe last patient's last assessment. It is anticipated that totalduration could be up to 24 weeks.

TABLE 19 12-Week Treatment Period 4-Week Safety Follow-up ScreeningVisit 2 Visit 3 Visit 6 Visit 8 Visit 1 Baseline Day Visit 4 Visit 5 Day84 Visit 7 Phone Call Day −56 Day 1 14 Day 28 Day 56 EOS Day 7⁶ Day 30Assessment to Day −1 (+/−1 day) (+4 days) (+/−3 days) Informed Consent XEligibility Review X X CAPS and Other X Medical History and DemographicsQuantiFERON X Gold Test for TB Height X Weight X Physical Exam¹ XLimited Physical X X X X X X Exam Physicians Global X X X X X X XAssessment of Disease Activity² Patient Diary X X X X X X X(daily)^(2, 8) Key Symptom X X X X X X X Score^(2, 8) HIV and Hep XScreen SARS-CoV-2 X Testing by RT- PCR Vital Signs^(2, 3) X X X X X X X12-Lead ECG^(2, 4) X X X Hematology, X X X X X X X Coagulation,Chemistry, Lipids, and Urinalysis PD Blood X X X X X X Sampling² PKBlood X X X X X X Sampling² Serum Pregnancy X Urine Pregnancy X X X X XX Dispense Study X X X X Medication Administration of X X X X X MorningDose of ATI-450 in Clinic⁷ Drug X X X X Accountability Adverse Events⁵ XX X X X X X X Prior and X X X X X X X X Concomitant Medications ECG =electrocardiogram, Hep = hepatitis, PD = pharmacodynamic, TB =tuberculosis, EOS = end of study ¹A full physical examination will beperformed at screening. The brief physical examination (including signsof CAPS) will be performed at visit 6 and follow-up. Body mass indexwill be derived in the eCRF. ²On dosing day(s), patient diary, PGA, KSS,vitals, clinical laboratory parameters, PK, and PD will be performedbefore the administration of study medication. ³ Vital signs will bemeasured prior to dosing and immediately following dosing in a semisupine position after 5 minutes' rest and will include temperature,systolic and diastolic blood pressure, pulse, and respiratory rate. ⁴ Atriplicate 12-lead ECG will be recorded after the subject has beenresting for at least 5 minutes in a supine position. ⁵Serious adverseevent reporting will start at the time of consent. Any AE that occursbetween the time of consent and dosing on Day 1 will be recorded asmedical history. Treatment-emergent AEs will be collected following thefirst dose of study medication on Day 1. ⁶Patients should not starttheir anti-IL-1 therapy until all safety follow-up Day 7 assessmentshave been completed. However, in the event of a safety concern, patientswill be allowed to return early or restart their anti-IL-1 therapy earlyprior to completing the safety follow-up Day 7 visit at the discretionof the investigator. ⁷The last dose of ATI-450 will be administered inclinic on Day 84. The second dose of ATI-450 is not to be administeredon Day 84. ⁸ Patients will be instructed on how to complete theirpatient diary at the screening visit. Patients will begin completing thepatient diary 7 days prior to beginning ATI-450 administration (Day 1),or immediately beginning at the screening visit if the Day 1 visit isscheduled to occur less than 7 days after screening.

Statistical Methods:

Determination of Sample Size—The sample size for this study wasdetermined based upon feasibility constraints as opposed to a formalpower computation. Up to 10 patients are planned to be enrolled.

Analysis Populations. The Intent-to-treat (ITT) population will includeall patients who have been administered at least one dose of studymedication. The Per-Protocol (PP) population will include all patientswho complete 12-weeks of treatment and 4-weeks of safety follow-up andhave completed study assessments.

Efficacy Analyses—All efficacy summaries will be conducted on both theITT and PP populations.

-   -   Proportion of participants in disease remission over time; where        disease remission is defined as having a PGA score of minimal or        better and a hsCRP and SAA value within the normal range or        within 30 percent of the baseline value (ITT and PP population).    -   Proportion of participants in clinical remission over time;        where clinical remission is defined as having a PGA score of        minimal or better (ITT and PP population).    -   Proportion of participants who experience an increase over        baseline in daily KSS of ≥3 for at least two days after        discontinuation of ATI-450 over time (PP population).    -   Proportion of participants who maintain a mean KSS of no more        than 2 points higher than baseline in at least 6 out of 8 weeks        in the ITT and PP population.    -   Change from baseline in PGA and KSS scores will be reported        descriptively over time (ITT and PP population).    -   Change from baseline in hsCRP and SAA and percent change from        baseline in hsCRP and SAA will be summarized over time using        continuous statistical summary measures. Determination of        sustained treatment effect for hsCRP and SAA will be based upon        the median percent change from baseline in the ITT and PP        population.

Efficacy Assessments

Physician Completed Efficacy Questionnaire. The Physician's GlobalAssessment of Autoinflammatory Disease Activity (PGA) is a measure to becompleted by the investigator or designee. The PGA uses a 5-point ratingscale: absent, minimal, mild, moderate, and severe. The investigatorwill select a rating based on the patient's current disease activity atthe time of the visit.

Maintenance of Disease Remission and Clinical Remission. Maintenance ofdisease remission will be assessed during the treatment period. Diseaseremission is defined as having a PGA score of absent or minimal and ahsCRP and SAA value within the normal range (≤10 mg/L) or within 30percent of the baseline value.

Maintenance of clinical remission will be assessed during the treatmentperiod. Clinical remission is defined as having a PGA score of absent orminimal.

Time to Relapse Time to relapse will be assessed during the treatmentperiod. Relapse is defined as a two-point worsening on the PGA scale.

Patient Completed Efficacy Questionnaires. When these assessments arerequired at the times outlined in Table 13, they should be the firsttasks done at any visit, and prior to study medication dosing.

The Key Symptom Score (KSS) is derived from the patient-administereddaily health assessment form (DHAF), and is the average on a 0 to 10scale (0=None, 10=Very Severe) of 5 separate scales—rash, feeling offever and chills, joint pain, eye redness and pain, and fatigue.

The DHAF is designed using a linear rating scale of circles in half-stepunits (e.g., 0.5, 1.0, 1.5, 2.0, etc.), which are marked 0 (none, noseverity) to 10 (very severe). Patients will select the circle on thescale that they determine most accurately represents severity of thesymptom that they have experienced during the last 24 hours.

The DHAF will be completed daily by the patient. Daily KSS will becalculated by averaging the sum of the 5 individual symptom scores. KSSduring a specified time period will further be calculated by averagingthe sum of daily KSS scores during the time period.

Patients will be provided a quiet, private place to complete theassessments. Patients will be instructed to answer all the questions tothe best of their ability and without help from others (study staff,family, or friends). The study staff should review the questionnairesafter they are completed and encourage patients to complete any missinginformation. Patients can refrain from answering any question. Studystaff will record the refusal of patients to answer any question in thesource documents.

Re-emergence of CAPS Symptoms Re-emergence of CAPS symptoms will beassessed after ATI-450 is discontinued following completion of thetreatment period. Re-emergence is defined as an increase in daily KSS of≥3 from baseline for at least two consecutive days.

High Sensitivity C-reactive Protein and Serum Amyloid A. Blood samplesfor evaluation of hsCRP and SAA will be collected at the times specifiedin Table 13. Samples will be shipped to a central laboratory. Specificinstructions for collection, processing, storage and shipment of bloodsamples will be provided in a separate laboratory manual. The normalrange for serum hsCRP and SAA is defined as ≤10mg/L for both.

Safety Assessments

Adverse Events: AEs will be followed, recorded, and reported.

Clinical Laboratory Evaluations: Laboratory assessments during treatmentwill be performed by a central laboratory. Blood and urine samples willbe collected at the times indicated in Table 13. On dosing day(s),sampling for the analysis of clinical laboratory parameters will beperformed before the administration of study medication.

Unless indicated otherwise, all laboratory samples will be processed andshipped to the central laboratory, as described in the centrallaboratory manual. The central laboratory will analyze the samples orsend them to reference laboratory(ies) for analysis, as indicated in themanual. Refer to the central laboratory manual for the maximum totalvolume of blood to be collected per patient throughout the study.

The following parameters will be assessed:

Hematology: hemoglobin, hematocrit, red blood cells, platelets, totalWBC count, differential WBC count, and ANC

Coagulation: INR, partial thromboplastin time, and prothrombin time

Biochemistry: albumin, alkaline phosphatase (ALP), ALT, amylase, AST,blood urea nitrogen (BUN), calcium, creatine phosphokinase, hsCRP,creatinine, gamma glutamyltransferase, glucose, inorganic phosphatase,lactate dehydrogenase, lipase, magnesium, potassium, SAA, sodium,chloride, bicarbonate, total bilirubin, total protein, and uric acid

Lipids: total cholesterol, high-density lipoprotein, low-densitylipoprotein, and triglycerides

Urinalysis: pH, specific gravity, creatinine, glucose, bilirubin, blood,and protein

Potential Drug-induced Liver Injury. Hy's Law cases have the following 3components: (1) The drug causes hepatocellular injury, generally shownby a higher incidence of ≥3-fold elevations above the ULN of ALT or ASTthan the placebo; (2) Among study patients showing such aminotransferaseelevations, often with aminotransferases much greater than 3× ULN, oneor more also shows elevation of serum total bilirubin to >2× ULN orINR>1.5, without initial findings of cholestasis (elevated ALP); (3) Noother reason can be found to explain the combination of increasedaminotransferase and total bilirubin, such as viral hepatitis A, B, orC; evidence for biliary obstruction; acute alcoholic hepatitis (recentdrinking and AST >2× ALT are supportive); recent history of severehypotension or congestive heart failure; other underlying viral disease;pre-existing or acute liver disease; or another drug (includingnon-prescription products such as herbal supplements) capable of causingthe observed injury

Example 9 ATI-450 Inhibits Inflammatory Cytokines Involved in AutoimmuneConditions

ATI-450 Increases Regulatory T (Treg) Cells in Mouse Collagen-InducedArthritis Model: To investigate the utility of ATI-450 for the treatmentof autoimmune conditions, the effect of ATI-450 on T cell subsets wasevaluated in a murine model of collagen-induced arthritis (CIA). In thismodel DBA/1 mice (12/group) were immunized with bovine collagen/CFA toinduce arthritis. At day 18, animals were administered vehicle, ENBREL(10 mg/kg QD), or ATI-450 (1000 ppm chow). At day 21, mice were boostedwith bovine collagen/CFA, and at day 35 the study was terminated.Popliteal lymph nodes were dissected from mice, cleaned of any tracefat, mechanically dissociated and filtered through 70 micron cellstrainers. Live lymphocytes were counted using a Moxi GO II cell counter(Orflo). One million lymphocytes were plated, treated with Fc block, andstained with antibodies targeting surface receptors (including CD45,CD3, CD4) along with a fixable live/dead dye. Cells werefixed/permeabilized (eBioscience Transcription Factor Staining kit) andstained with anti-Foxp3 antibody. The cells were collected via flowcytometry analysis on an Attune NxT instrument. T-regulatory cells weregated as lymphocyte-sized, live, CD45+ CD3+ CD4+ Foxp3+ cells.

As shown in FIG. 21, ATI-450 treatment in the murine CIA modelsignificantly increased the number of regulatory T cells (TREGs). GivenTREG cells known involvement in the suppressing the immune response andprevention of autoimmune disease, this data indicates the utility ofATI-450 for the treatment of autoimmune conditions, such as inflammatorybowel disease, systemic lupus nephritis (SLE), and others.

ATI-450 blocks Lipopolysaccharide (LPS) stimulated TNF-α, IL-1β and IL-6production in Human Whole Blood. Venous blood from healthy humanvolunteers was collected in tubes containing sodium heparin andaliquoted (180 μL per well) into 96 well round-bottom tissue cultureplates. Compounds were serially diluted in 100% DMSO (Sigma-Aldrich,D2650) followed by a 50× dilution into DMEM/10% FBS (Gibco,11965/26140). The diluted compounds were then added to each plate induplicate 10 μL aliquots for final concentrations as indicated in FIG.22A. Vehicle consisted of DMSO at a final assay concentration of 0.1%.Pin tools (V&P Scientific, Inc., VP246) were placed in the wells and theplate was placed at 37° C. with 5% CO₂, with gently shaking for fiveminutes, then pin tools were removed and saved. After a one-hourincubation at 37° C. with 5% CO₂, 10 μL of LPS (Sigma-Aldrich, L2630),in DMEM/10% FBS (Gibco, 11965/26140) was added for a final concentrationof 100 ng/mL to each well except for the no stimulus wells. Pin toolswere placed in the wells in the same orientation as the previous stepand gently shook for five minutes at 37° C. with 5% CO₂, then removed.After five hours of incubation with LPS at 37° C. with 5% CO₂, eachreaction plate of blood was centrifuged at 1,800×g for five minutes. Theplasma was harvested and assayed for TNF-α, IL-1β, and IL-6 using MesoScale Discovery Technology V-plex human cytokine kits according to thekit protocol. The kit plates were read using an MSD Sector S 6000instrument. Calculations to establish cytokine calibration curves and todetermine analyte concentrations in the samples were carried out usingthe MSD DISCOVERY WORKBENCH® analysis software.

LPS stimulates Toll-like receptor 4 (TLR4) release of a number ofpro-inflammatory cytokines. As show in FIG. 22A, ATI-450 potentlyinhibited LPS-stimulated IL-6, TNF-α, and IL-1β with IC₅₀ values ofbetween 0.01-0.1 μM. While ATI-450 mediated inhibition of TLR4stimulated cytokine release was expected given ATI-450's role MK2inhibition, it was unknown whether ATI-450 would similarly blockcytokine release triggered by other stimuli, such as TLR3 and TLR 7,8activation.

ATI-450 Inhibited Poly(I:C) stimulated IP10, IFNγ, IL-6, IL-8 and TNF-αproduction in Human Whole Blood. Human whole blood assays were performedas described above with the exceptions listed below. Human whole bloodwas aliquoted (175 μL per well) into 96 well round-bottom tissue cultureplates. Compounds were serially diluted in 100% DMSO (Sigma-Aldrich,D2650) followed by a 25× dilution into DMEM/10% FBS (Gibco,11965/26140). The diluted compounds were then added to each plate induplicate 5 μL aliquots for final concentrations as indicated in FIG.22B. After the one-hour incubation with the compounds or vehicle, 20 μLof poly(I:C) (InvivoGen, HMW, tlrl-pic), in DPBS (Gibco, 14190-136) wasadded for a final concentration of 100 μg/mL to each well except for theno stimulus wells. After twenty-four hours of incubation with poly(I:C),plasma was harvested and assayed for IP10, IFNγ, IL-6, IL-8 and TNF-αusing Meso Scale Discovery Technology V-plex human cytokine kitsaccording to the kit protocol.

Poly(I:C) is a TLR3 agonist that induces the activation of theproinflammatory cytokines, IL-6, TNF-α, interferon-y and theinflammatory chemokines, IL-8 and interferon-y inducible protein (IP10).As shown in FIG. 22B, ATI-450 was able to potently inhibit in vitro TLR3stimulated cytokines and chemokines with IC₅₀ values of between 0.01-0.1μM.

ATI-450 Inhibited R848 stimulated TNF-α, IL-1β, IL-6 and IL-8 productionin Human Whole Blood. Human whole blood assays were performed asdescribed above with the exceptions listed below. Human whole blood wasaliquoted (200 μL per well) into 96 well round-bottom tissue cultureplates. Compounds were serially diluted in 100% DMSO (Sigma-Aldrich,D2650) followed by a 10× dilution into RPMI (Gibco, 11875). The dilutedcompounds were then added to each plate in duplicate 2 μL aliquots forfinal concentrations as indicated in FIG. 22C. After the one-hourincubation with the compounds or vehicle, 2 μL of R848 (InvivoGen,tlrl-r848) in RPMI (Gibco, 11875) was added for a final concentration of0.5 μg/mL to each well except for the no stimulus wells. After fivehours of incubation with R848, plasma was harvested and assayed forTNF-α, IL-1β, IL-6 and IL-8 using Meso Scale Discovery Technology V-plexhuman cytokine kits according to the kit protocol.

R848 (resiquimod) is a potent agonist of TLR 7 and TLR8. TLR7 and TLR8activation induces expression and activity of various inflammatorycytokines and chemokines including IL-6, IL-8, TNF-α, and IL-1β. Asshown in FIG. 22C, ATI-450 was able to potently inhibit in vitro TLR7-and TLR8-stimulated cytokines and chemokines with IC₅₀ values of ˜0.1μM.

While ATI-450 mediated inhibition of TLR4 stimulated cytokine releasewas expected given ATI-450's role in MK2 inhibition of pro-inflammatorysignaling pathways, it was unexpected that ATI-450 would similarly blockcytokine release triggered by multiple other stimuli, such as TLR3 andTLR7 and TLR8 receptor activation. These data indicated that ATI-450 hastherapeutic utility for the treatment of autoimmune disease and otherdisorder that have a strong anti-nuclear antibody drive, e.g., lupus,scleroderma, Sjogren's syndrome, juvenile arthritis, etc.

To further assess ATI-450 inhibition of various cellular cytokines,multiple cell types and various stimuli were tested as described below.The results of these studies are presented in Table 14 below.

U937 Cell differentiation and LPS stimulated cytokine assay: The U937human pre-monocytic cell line (ATCC, CRL-1593.2) was grown in completemedia: RPMI 1640 (Gibco, A10491) with penicillin-streptomycin (10U/mL)-glutamine (2 mM) (Gibco, 10378016) and 10% FBS(Gibco, 26140-079).Cells were differentiated to a monocyte/macrophage phenotype with theaddition of PMA (Sigma-Aldrich, P1585) (20 ng/mL, 24 hours), washed withDPBS (Gibco, 14190-136) and incubated in complete media for 24 hours at37° C., 5% CO₂. Following recovery, the cells were scraped, counted,re-plated in complete media in accordance with the experimental designand incubated for an additional 24 hours at 37° C., 5% CO₂ prior tostimulation with LPS (Sigma-Aldrich, L2630) as described below.

Differentiated U937 cells were pretreated for one hour in the presenceof serially diluted compound or vehicle then stimulated with LPS at afinal concentration of 100 ng/mL for four hours. Culture media was thencollected for determination of cytokine levels using Meso ScaleTechnology.

A549 IL-1β stimulated IL-6 Assay—A549 cells (American Type CultureCollection, ATCC CRL-1593.2) were grown in Ham's F-12K (Kaighn's)(Gibco, 21127-022) with penicillin-streptomycin (10 U/mL) (Gibco,15140-122) and 10% fetal bovine serum (Gibco, 26140-079). Cells weregrown to 80% confluence then trypsinized to remove cells. Cells wereplated in 96 well flat bottom plates in complete media and allowed torecover overnight. Cells were pretreated for one hour in the presence ofserially diluted compound or vehicle (0.5% DMSO) (Sigma-Aldrich, D2650)then stimulated with IL-1β (1 ng/mL) (R&D Systems, 201-LB-005) for 18hours. Culture media was then collected for determination of IL-6 levelsusing Meso Scale Technology.

Human PBMC LPS Stimulated IL-1β Assay—Frozen Human PBMC stocks wererapidly thawed, resuspended in DMEM/10% FBS (Gibco, 11965/26140),pelleted at 130×g for 5 minutes then resuspended in DMEM/10% FBS andplated at 200,000 cells per well (180μ1) in flat bottom 96 well plates.The cells were allowed to recover for several hours at 37° C. with 5%CO₂. Compounds were serially diluted in 100% DMSO (Sigma-Aldrich, D2650)followed by a 50× dilution into DMEM/10% FBS (Gibco, 11965/26140). Thediluted compounds were then added to each plate in duplicate 10 μLaliquots. Vehicle consisted of DMSO at a final assay concentration of0.1%. After a one-hour incubation at 37° C. with 5% CO₂, 10 μL of LPS(Sigma-Aldrich, L2630), in DMEM/10% FBS (Gibco, 11965/26140) was addedfor a final concentration of 100 ng/mL to each well except for the nostimulus wells. After twenty-four hours of incubation with LPS at 37° C.with 5% CO₂, each reaction plate of PBMC was centrifuged at 130×g forfive minutes. The supernatant/conditioned media was harvested andassayed for IL-1β using Meso Scale Discovery Technology V-plex humancytokine kits according to the kit protocol.

Porcine LPS stimulated TNF-α Assay—Porcine whole blood was collectedinto heparinized vacutainers then transferred into conical tubes andshipped overnight on wet ice. Porcine whole blood was aliquoted (160μL/well) into round bottom 96 well tissue culture plates. Compounds wereserially diluted in 100% DMSO (Sigma-Aldrich, D2650) followed by a 100×dilution into RPMI/10% FBS (Gibco, 11875/26140). The diluted compoundswere then added to each plate in duplicate 20 μL aliquots for finalconcentrations ranging from 10 μM to 1 pM. DMSO final concentration was0.1%. After a one-hour incubation at 37° C. with 5% CO₂, 20 μL of LPS(Sigma-Aldrich, L2630), in RPMI/10% FBS (Gibco, 11875/26140) was addedfor a final concentration of 10 μg/mL to each well except for the nostimulus wells. After four hours of incubation with LPS, plasma washarvested by centrifugation at 1,800×g for five minutes. The plasmaswere assayed for TNF-α levels using a porcine specific TNF-α QuantikineELISA kit (R&D, PTA00) according to the kit protocol.

TABLE 20 IC₅₀ (nM, Mean ± Standard Deviation) Cell System StimulantTNF-α IL-1α/β IL-6 U937 cells LPS, 100 ng/mL, 4 hr  1.55 ± 0.35 A549cells IL-1β, 1 ng/mL, 18 hr 11.1 ± 7.6 hPBMC LPS, 100 ng/mL, 24 hr 31.1(IL-1α) mPMN IL-1α, 10 ng/mL, 4 hr 5.0 Rat Whole Blood LPS, 100 ng/mL, 4hr 14.3 ± 1.9 28.3 ± 1.2 Human Whole Blood LPS, 100 ng/mL, 4 or 24 hr29.0 ± 9.0 6.1 (IL-1β) 135 ± 97 Human Whole Blood IL-1β, 10 ng/mL, 5 hr12.2 ± 4.0 11.1 ± 3.8 Human Whole Blood R848, 0.5 ug/mL, 5 hr 34.8 ± 5.095.6 ± 34.8 (IL-1β)  79.2 ± 17.2 Human Whole Blood Poly I:C, 100 ug/mL,24 hr  20.8 ± 12.8  13.2 ± 12.8 Pig Whole Blood LPS, 10 mg/mL, 4 hr 7.4

Example 10 ATI-450 Inhibits Cytokines Involved in Inflammatory SkinConditions

ATI-450 Inhibits TLR2 Stimulated Cytokine Signaling in vitro:Significant literature suggest that TLR2 agonists drive TH17differentiation and suppress regulatory T cells (see e.g., Nyirenda etal., “TLR2 Stimulation Drives Human Naive and Effector Regulatory TCells into a Th17-Like Phenotype with Reduced Suppressive Function,” J.Immunol. 187(5):2278-90 (2021), which is hereby incorporated byreference in its entirety). To investigate whether ATI-450 mediatedinduction of Treg cells involved inhibition of TLR2 agonist signaling,the ability of ATI-450 to inhibit LPS or PAM3 (TLR2 agonist) TNF-αlevels in vitro was examined. To this end, peripheral blood mononuclearcells were pre-incubated for 1 hour with ATI-450 at final concentrationsof 10-10,000 nM (or DMSO to give a final of 0.1% in all wells). LPS orPam3 were then added to the designated wells for a final concentrationof 100 ng/mL for both (or medium for unstimulated control). After 30 minMSU was added to the designated wells for final concentration of 100μg/mL (or medium for no-MSU control). After a 6-hour incubation, plateswere centrifuged at 500×g for 5 min, and medium was collected from eachwell for assay.

Released TNF-α was measured using Meso Scale Discovery technology, anELISA-like format with capture and detection antibodies, but with afinal readout using electrochemiluminescence.

As shown in FIG. 23, ATI-450 dose dependently inhibits TLR2 ligandinduced TNF-α production. As noted above TLR2 signaling drives TH17 celldifferentiation.

ATI-450 Inhibits IL-17 Production in vitro: To investigate whetherATI-450 mediated inhibition of the TLR2 pathway also plays a role inmodulating IL-17 production, total CD4+ cells were isolated from humanPBMCs using the CD4 StemCell Isolation Kit. CD4+ cells were cultured for3 days under TH17 skewing conditions (anti-CD3, anti-CD28, anti-IL-4,anti-IFNγ, rIL-6, rIL-23, rTGFβ). On day 3, cells were harvested,washed, re-plated, and treated with compound or DMSO only. After 1 hrincubation, cells were re-stimulated overnight with anti-CD3 dynabeadsat a 1:1 cell:bead ratio. After 18-20 hours, supernatants were collectedand assayed for IL-17A levels using MSD. As shown in FIG. 24, ATI-450partially inhibits IL-17 production in CD4+ cells.

Inhibition of Il-17 has demonstrated utility in the treatment ofpsoriasis, psoriatic arthritis, and spondylitis. The observed partialinhibition of IL-17 by ATI-450 may also render ATI-450 particularsuitable for the treatment of IBD, where only partial IL-17 inhibitionis thought to provide efficacy without exacerbating IBD.

ATI-450 Inhibits IL-1β stimulated TNF-α, IL-6 and IL-8 production inHuman Whole Blood—Human whole blood assays were performed as describedabove with the exceptions listed below. Human whole blood was aliquoted(200 μL per well) into 96 well round-bottom tissue culture plates.Compounds were serially diluted in 100% DMSO (Sigma-Aldrich, D2650)followed by a 10× dilution into RPMI (Gibco, 11875). The dilutedcompounds were then added to each plate in duplicate 2 μL aliquots forfinal concentrations as indicated in FIG. 25. After the one-hourincubation with the compounds or vehicle, 2 μL of IL-1β (R&D,201-LB-010) in RPMI (Gibco, 11875) was added for a final concentrationof 0.5 μg/mL to each well except for the no stimulus wells. After sixhours of incubation with IL-1β, plasma was harvested and assayed forTNF-α, IL-6 and IL-8 using Meso Scale Discovery Technology V-plex humancytokine kits according to the kit protocol.

As shown in FIG. 25, ATI-450 dose dependently inhibits IL-1β-stimulatedTNF-α (IC₈₀ 31±6 ng/mL), IL-6 (IC₈₀ 41±20 ng/mL), and for IL-8 (IC₈₀40±12 ng/mL) in human whole blood.

ATI-450 Inhibits IL-1α biosynthesis and activity in human immune cells.Human PBMCs were pre-treated with ATI-450 for 1 hour, and thenstimulated with LP for 24 hours. Biosynthesis of IL-1α was quantifiedusing MSD technology. In a separate study, mouse neutrophils wereisolated from CD-1 mouse bone marrow, plated, and rested for one hourprior to addition of various concentrations of ATI-450 which was addedand incubated for 1 hour at 37° C. Cells and compound were stimulatedwith mouse IL-1α to a final concentration of 10 ng/mL and incubated for4 hours at 37° C. Supernatants were collected and TNF-α levelsquantitated using MSD technology.

As shown in FIG. 26A, ATI-450 dose dependently inhibited LPS inducedIL-1α biosynthesis, with an IC₅₀ value of 31.1 nM. ATI-450 also dosedependently inhibits IL-1α mediated TNF-α signaling in neutrophils asshown in FIG. 26B.

Example 11 Formulations of ATI-450 Tablets

The compositions of the tablets comprising ATI-450 and tabletscomprising placebo used in the Examples herein are provided in Table 21below. The excipients used in the drug tables and their function areprovided in Table 22 below.

TABLE 21 Composition of ATI-450 tablets 10 mg Strength Placebo 50 mgStrength Placebo Component % w/w mg/tab % w/w mg/tab % w/w mg/tab % w/wmg/tab ATI-450 12.5 10 — — 12.5 50 — — Silicified 71 56.8 83.5 66.8 71284 83.5 334 microcrystalline cellulose Mannitol 10 8 10 8 10 40 10 40Crospovidone 5 4 5 4 5 20 5 20 Hydrophilic fumed 0.75 0.6 0.75 0.6 0.753 0.75 3 silica Magnesium stearate 0.75 0.6 0.75 0.6 0.75 3 0.75 3 Total100 80 100 80 100 400 100 400

TABLE 22 Excipients in Tablets Comprising ATI-450 Quality ExcipientChemical Name Function Standard ProSolv HD 90 Silicified Filler/BinderNF/EP/JP microcrystalline cellulose Perlitol 200 SD Mannitol DiluentUSP/EP Polyplasdone XL Crospovidone Disintegrant NF/EP/JP Aerosil 200Colloidal silicone Glidant NF/EP/JP dioxide Magnesium stearate Magnesiumstearate Lubricant NF/EP/JP

Example 12 Separation of Compound (P)-I

Racemic3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-2′-(2-(2-hydroxypropan-2-yl)pyrimidin-4-yl)-5′,6-dimethyl-2H-[1,4′-bipyridin]-2-one(250 mg, 0.49 mmol) may be prepared according to the methods describedin U.S. Pat. No. 9,115,089, which is hereby incorporated by reference inits entirety. Chiral resolution of3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-2′-(2-(2-hydroxypropan-2-yl)pyrimidin-4-yl)-5′,6-dimethyl-2H-[1,4′-bipyridin]-2-oneto obtain the P atropisomer, i.e., Compound (P)-I as disclosed herein,is carried out as described below.

Racemic3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-2′-(2-(2-hydroxypropan-2-yl)pyrimidin-4-yl)-5′,6-dimethyl-2H-[1,4′-bipyridin]-2-one(250 mg, 0.49 mmol) was separated using supercritical fluidchromatography (Thar 80, preparative SFC, ChiralCel OD-H, 250×30 mm IDcolumn) with a mobile phase of carbon dioxide and ethanol. Theseparation method used an isocratic method of 40% ethanol with a flowrate of 50 mL/min and a cycle time of 10 min. Optical rotation wasdetermined using a WZZ-2S polarimeter.

The faster isomer ((P)-I) eluted at 1.77 minutes yielded 115 mg of(−)-3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-2′-(2-(2-hydroxypropan-2-yl)pyrimidin-4-yl)-5′,6-dimethyl-2H-[1,4′-bipyridin]-2-onein ethylene glycol: [α]_(D) ²⁰ −46° (CH₃OH); ¹H NMR (400 MHz, DMSO-d₆) δppm 8.97 (d, J=5.09 Hz, 1H), 8.86 (s, 1H), 8.69 (s, 1H), 8.61 (s, 1H),8.24 (d, J=5.08 Hz, 1H), 8.10 (t, 1H), 6.85 (s, 1H), 5.50 (s, 2H), 5.26(s, 1H), 2.11 (s, 3H), 1.98 (s, 3H), 1.54 (s, 3H), 1.52 (s, 3H); MS (ES)m/e 514 (M+H).

The slower isomer ((M)-I) eluted at 3.68 minutes yielded 112 mg of(+)-3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-2′-(2-(2-hydroxypropan-2-yl)pyrimidin-4-yl)-5′,6-dimethyl-2H-[1,4′-bipyridin]-2-onein ethylene glycol: [α]_(D) ²⁰ +45° (CH₃OH); ¹H NMR (400 MHz, DMSO-d₆) δppm 8.97 (d, J=5.09 Hz, 1H), 8.86 (s, 1H), 8.69 (s, 1H), 8.61 (s, 1H),8.24 (d, J=5.08 Hz, 1H), 8.10 (t, 1H), 6.85 (s, 1H), 5.50 (s, 2H), 5.26(s, 1H), 2.11 (s, 3H), 1.98 (s, 3H), 1.54 (s, 3H), 1.52 (s, 3H); MS (ES)m/e 514 (M+H).

Example 13 Crystal Form Screen of Compound (P)-I

The crystal-form screening study involved a total of 48 neat and binarysolvent systems which addressed the moderate solubility of the inputmaterial and provided a diverse set of polarities, dielectric constants,dipole moments, and hydrogen-bond donor/acceptor attributes.Water-containing solvents with a variety of water activities (a_(w))¹were also included to probe for the formation of hydrates. Temperaturesranging between 40° C. to −20° C.

The screening studies were comprised of the following crystallizationmodes:

-   -   Temperature-cycled ripening of API slurries between 5-40° C. for        four days (TC)    -   Rapid cooling clarified saturated solutions from 40 to −20° C.        and holding at −20° C. for three days (RC)    -   Slow evaporation of clarified solutions at RT over 14 days.        Rapid evaporation of solvents under reduced pressure from        solutions that did not produce solids during slow evaporation        after 14 days (EV).

A summary of the outcomes of the screening study are shown in Table 23.

TABLE 23 Results of the Crystal Form Screen Water # Solvent TC RC EVActivity 1 Water Form A 1.00 2 Methanol Form A 32-Methoxyethanol:Isopropyl Form A Form A ether (20:80) 4 1-Propanol FormA 5 Nitromethane Form A Form A Form A 6 Acetonitrile Form A Form A FormA 7 DMSO:t-butyl methyl ether Form A (20:80) 8 Acetone Form A 92-Butanone Form A 10 Dichloromethane Form A 11 Methyl acetate:HeptaneForm A (20:80) 12 4-Methyl-2-pentanone Form A Form A 13 Chloroform 14Ethyl acetate Form A 15 Chlorobenzene:Cyclohexane Form A (20:80) 16Tetrahydrofuran Form A 17 1,4-Dioxane Form A 18 Isopropyl ether Form A19 Toluene Form A Form A 20 Cyclohexane Form A 21 Heptane Form A 221-Butanol Form A 23 2-Propanol Form A Form A 24Trifluoroethanol:Isopropyl Form A ether (20:80) 25 Dimethyl carbonateForm A Form A 26 t-Butyl methyl ether Form A 27 Isopropyl acetate Form AForm A 28 Ethanol Form A 29 1-Methoxy-2- Form A propanol:Isopropyl ether(20:80) 30 Cyclohexanone 31 N,N- Form A 0.95 Dimethylformamide:Water(20:80) 32 2-Methoxyethyl Form A ether:Heptane (20:80) 33 Methanol:Water(95:5) Form A Form A 0.20 34 Acetonitrile:Water (95:5) Form A 0.94 35Acetone:Water (20:80) Form A Form A 0.96 36 Tetrahydrofuran::Water FormA Form A 0.82 (20:80) 37 2-propanol:Water (95:5) Form A Form A 0.55 38Methanol:Water (90:10) Form A Form A Form A 0.33 39 Acetonitrile:Water(90:10) Form A Form A 0.76 40 Acetone:Water (90:10) Form A 0.70 41Tetrahydrofuran:Water Form A 0.83 (90:10) 42 1,4-Dioxane:Water (90:10)Form A 0.70 43 2-propanol:Water (90:10) Form A Form A 0.65 44Acetone:Water (80:20) Form A Form A 0.77 45 Ethanol:Water (20:80) Form A0.93 46 Ethyl Form A acetate:cyclohexane (20:80) 47Acetonitrile:isopropyl Form A ethyl ether (20:80) 48 4-Methyl-2- Form Apentanone:heptane (20:80)

Example 14 Single Crystal Structure Determination of Compound (P)-I(Form A)

The crystalline form of Compound (P)-I has been characterized relativeto the absolute stereochemical configuration of the spatial arrangementof the atoms using single crystal X-ray diffraction. A detaileddescription of structure determination by X-ray diffraction is providedin Stout & Jensen, X-Ray Structure Determination: A Practical Guide,Macmillan Co., New York (1968), Chapter 3, which is herein incorporatedby reference. Alternatively, the unique spatial arrangements of atoms inthree dimensions within the crystalline lattice may be characterized byX-ray powder diffraction analysis. A detailed description of X-raypowder diffraction is provided in Cullity, B. D. Elements of X-rayDiffraction. Addison-Wesley, (1978) ISBN 0-201-01174-3 Chapter 14),which is herein incorporated by reference. PXRD data consists ofexperimentally determined values of the two-theta position, theintensity values of multiple crystallographic reflections, also known asBragg reflections, and their peak shape. The PXRD data may be analyzedcomputational, including by the method of Rietveld refinement. Adetailed description of Rietveld refinement of X-ray powder diffractiondata is provided in Pecharsky, Vitalij K.; Zavalij, Peter Y. (2009)Fundamentals of powder diffraction and structural characterization ofmaterials (2nd ed.). New York: Springer. ISBN 978-0-387-09579-0. OCLC314182615, which is herein incorporated by reference.

PXRD data may be collected at various temperatures or pressures in orderto facilitate Rietveld refinement. The experimental PXRD data including2-theta values, d-spacing, Bragg reflections and intensity values may becompared to a simulated PXRD pattern derived from the single crystalstructure determination which represents an idealized pure powder, usinga computational method such as described in Macrae, Clare F., et al.“Mercury 4.0: from visualization to analysis, design and prediction.”Journal of Applied Crystallography vol. 53, 226-235. 1 Feb. 2020,doi:10.1107/51600576719014092.

One of ordinary skill in the art will appreciate that an X-ray powderdiffraction pattern may be obtained with a measurement error that isdependent upon the measurement conditions employed in the datacollection. It is generally accepted that the peak shape, intensityvalues and two-theta positions derived from an X-ray powder diffractionpattern can fluctuate depending upon the type of instrument used, themeasurement conditions and the method of computational analysisperformed. It should be further understood that that the two-thetavalues and their relative intensities may also vary and accordingly, theexact order of intensity values should not be taken into account.

Additionally, the experimental error for diffraction angle measurementsfor a conventional X-ray powder diffraction pattern is typically about5% or less. Assessment of the extent of measurement error should betaken into account when describing the position of the two-thetadiffraction peaks. Consequently, it is to be understood that the crystalforms described in this invention are not limited to the crystal formsthat provide X-ray powder diffraction patterns completely identical tothe X-ray powder diffraction patterns depicted in the FIG. 27 disclosedherein. Any crystal forms that provide X-ray powder diffraction patternssubstantially identical to those disclosed in the accompanying Figuresfall within the scope of the present invention. The ability to ascertainsubstantial identities of X-ray diffraction patterns is within thepurview of one of ordinary skill in the art. Likewise, it is to beunderstood that any crystal forms that provide differential scanningcalorimetry (DSC) and/or thermogravimetric analysis (TGA) substantiallyidentical to those disclosed in the accompanying FIG. 28 fall within thescope of the present invention. The ability to ascertain substantialidentities of these patterns is within the purview of one of ordinaryskill in the art.

Crystalline Form A of Compound (P)-I is anhydrous and was obtained fromcrystallization conditions described in Example 13 utilizing variousorganic solvents and organic/water solvent systems.

X-Ray Powder Diffraction (PXRD) diffractograms were acquired onPANalytical X'Pert Pro diffractometer using Ni-filtered Cu Ka (45 kV/40mA) radiation and a step size of 0.02° 2θ and X'celerator™ RTMS (RealTime Multi-Strip) detector. Configuration on the incidental beam side:fixed divergence slit (0.25°), 0.04 rad Soller slits, anti-scatter slit(0.25°), and 10 mm beam mask. Configuration on the diffracted beam side:fixed divergence slit (0.25°) and 0.04 rad Soller slit. Samples weremounted flat on zero-background Si wafers.

Values of significant Bragg reflections, their 2-theta positions andd-spacing values, as compared to results from simulated PXRD data ofcrystalline Form A of Compound (P)-I are shown in Table 24 as derivedfrom the PXRD spectrum, shown in FIG. 27.

TABLE 24 Experimental and simulated PXRD data Experimental PXRDSimulated PXRD 2-Theta Bragg Reflections 2-Theta Angles (°) d (Å) h k 1Angles (°) d (Å) 5.21 16.946 0 0 1 5.20 17.2385 9.78 9.0362 0 1 0 9.808.99183 10.27 8.6059 0 1 1 10.26 8.48713 13.00 6.8073 0 1 2 13.166.92863 15.34 5.7705 0 1 −2 15.22 5.81934 15.51 5.7099 0 0 3 15.415.74618 16.92 5.2351 0 1 3 17.07 5.33869 17.92 4.9473 1 1 −2 17.914.94901 18.86 4.7017 1 −1 1 18.85 4.70444 19.60 4.5254 0 2 1 19.664.58616 20.57 4.3147 1 −1 −2 20.65 4.30016 21.01 4.2259 0 2 2 20.924.24356 23.60 3.7675 0 2 −2 23.58 3.77065 24.29 3.6608 0 1 4 24.133.65807 25.92 3.4341 2 2 2 25.70 3.46431 29.05 3.0712 1 −1 2 29.193.0598 29.48 3.0275 0 3 1 29.48 3.02715

Differential Scanning Calorimetry (DSC) was conducted with a TAInstruments Q100 differential scanning calorimeter equipped with an autosampler and a refrigerated cooling system under 40 mL/min N₂ purge. DSCthermograms were obtained at 15° C./min in crimped Al pans. FIG. 28depicts one such thermogram (bottom trace), obtained from a sample of(P)-I.

Thermogravimetric Analysis (TGA) thermograms were obtained with a TAInstruments Q500 thermogravimetric analyzer under 40 mL/min N₂ purge at15° C./min in Pt or Al pans.

DSC analysis, depicted in FIG. 28 (top trace) indicates crystalline FormA of Compound (P)-I exhibits a melting/racemization event at 187.92° C.,followed by a recrystallization event at 195.8° C., and finally a sharpendotherm at 253.5° C. (melt of racemate). Negligible weight loss (0.7%)is observed between 25° C. and 256° C. by TGA.

Fourier Transform Infrared Spectroscopy (FT-IR): Characteristic spectralabsorbance data from FT-IR of Form A of Compound (P)-I showing thelocation of significant IR-active regions and their functional groupassignments is shown in Table 25.

TABLE 25 FT-IR Absorbance Data Wavenumber (cm−1) Functional GroupAssignment 3486 —OH stretch 3072 C—H (aromatic) stretch 2982 C—H (CH3)stretch 1656 C═O stretch 1605 C═N stretch 1592 C═N stretch 1571 C═Cstretch 1546 C═N stretch 1525 C═C stretch 1476 C—H (CH2) bend 1457 CH2scissor 1429 C—H (CH3) bend 1385 C—H (gem dimethyl) bend 1380 —O—H bend1350 C—N(pyridone) stretch 1296 C—F stretch 1237 C—O (conjugated, alkylether) stretch 1214 C—H (aromatic) bend 1184 C—O (tertiary alcohol)stretch 1130 C—F stretch 1103 C—H (aromatic) bend 1051 C—F stretch 1044C—F stretch 1005 C—H (aromatic) bend 978 C—H (aromatic) bend 964 C—H(aromatic) bend 860 C—H (aromatic) bend 840 C—H (aromatic) bend 810 C—H(aromatic) bend 793 C—H (aromatic) bend 781 C—H (aromatic) bend 755 C—Clstretch 741 C—H (aromatic) bend 703 C—H (aromatic) bend 669 N—C═O bend

Example 15 p38 Inhibitory Potency and p38/MK2 Substrate Selectivity ofCompound (P)-I

Inhibition of MK2 and PRAK by Compound (P)-I and (M)-I, respectively,was investigated to understand selectivity of Compound (P)-I and/orCompound (M)-I in reducing inflammatory response. Compound (P)-I and(M)-I were evaluated in enzyme assays that compared inhibitor potency inblocking p38/MK2 versus p38/PRAK-induced phosphorylation of anHSP-27-derived peptide substrate. The ability of Compound (P)-I and(M)-I to inhibit activated phospho-p38α was evaluated using a p38α/MK2and a p38α/PRAK cascade assay format. The kinase activity of p38α wasdetermined by its ability to phosphorylate GST-MK2 or GST-PRAK.Activation of MK2 or PRAK by p38α was quantitated by measuring thephosphorylation of a fluorescently-labeled, MK2/PRAK specific peptidesubstrate, Hsp27 peptide. The phosphorylation of the Hsp27 peptide wasquantified using IMAP technology (Molecular Devices, Sunnyvale Calif.).Kinase reactions were carried out in a 384-well plate (Greiner, 781280)in 20 mM HEPES pH 7.5, 10 mM MgCl₂, 0.01% Triton X-100, 0.01% BSA, 1 mMDTT, and 2% DMSO. The concentration of inhibitor in the assays wasvaried between 0.02 nM to 30,000 nM, while the Hsp27 peptide substrateand MgATP were held constant at 1 μM and 10 μM, respectively. Activatedp38α was added to a final concentration of 30 pM for reactions withnon-phosphorylated 1 nM GST-MK2 in the cascade reaction. For thep38α/PRAK cascade, the concentration of non-activated GST-PRAK was heldconstant at 10 nM while p38α was added to a final concentration of 200pM. Kinase reactions were incubated at room temperature and quenchedafter 120 minutes by the addition of IMAP Binding Solution. Under theseconditions, approximately 20% of the substrate Hsp27 peptide wasphosphorylated. Reactions were initiated by the addition of activatedp38α except for pre-incubation experiments, where reactions wereinitiated by the addition of Hsp27 peptide and MgATP. Pre-incubation ofp38α with inhibitor or p38α with non-activated GST-MK2 or non-activatedGST-PRAK and inhibitor were performed at 2× final assay concentrationsat room temperature 240 minutes prior to adding ATP and Hsp27 peptide toinitiate catalysis. The inhibitory potency of Compounds (P)-I and (M)-Iwas quantitated from dose-response IC₅₀ values or K_(i) values fromp38α/MK2 cascade assays while the substrate selectivity was calculatedas a ratio of p38α/PRAK:p38α/MK2 IC₅₀ values.

Compound (P)-I and Compound (M)-I were tested in accordance with theabove described assay, yielding IC₅₀ values described in Table 26 below:

TABLE 26 p38/MK2 p38/PRAK Selectivity Compound Structure IC₅₀ (μM) IC₅₀(μM) Ratio (P)-I

0.021 8.1 385x (M)-I

3.48 12.5 3.6

Example 16 Cytokine Regulation in Human Monocytes by Compound (P)-I

The p38 pathway has been shown to be critical for the biosynthesis of anumber of pro-inflammatory cytokines including TNFα, IL-1β and IL-6.Therefore, inhibition of the p38 MAP Kinase pathway will lower theinflammatory response by decreasing biosynthesis of pro-inflammatorycytokines. This study shows the amount of Compound (P)-I and Compound(M)-I necessary to inhibit biosynthesis of TNFα, IL-6, and IL-1β(pro-inflammatory cytokines) by half, demonstrating the effectiveness ofeach compound in reducing inflammation. Evaluation of the potency andefficacy of Compound (P)-I and Compound (M)-I to block cytokineproduction were carried out using the human U937 cell line. The U937human pre-monocytic cell line was obtained from the American TypeCulture Collection (Rockville, Md.). These cells were differentiated toa monocytic/macrophage phenotype as described by Burnette (Burnette etal, (2009). SD0006: a potent, selective and orally available inhibitorof p38 MAP Kinase, Pharmacology 84(1):42-60). Differentiated U937 cells(human peripheral blood mononuclear cells (hPBMC)) were seeded into96-well tissue culture plates (200,000 cells/well) in complete media.After 24 hours, the cells were pretreated for 60 minutes in the presenceor absence of Compound (P)-I and Compound (M)-I and then stimulated withLPS (0.1 μg/mL) for 4 hours. Culture media was then collected fordetermination of TNFα, IL-6 or IL-10 levels by ELISA. Cytokineconcentrations were extrapolated from recombinant protein standardcurves using a four-parameter logistic model and solving for IC₅₀ afteriterating to the best least-squares fit.

Both atropisomers of ATI-450 (Compounds (M)-I and (P)-I) were tested inaccordance with the above described assay, yielding IC₅₀ valuesdescribed in Table 27 below:

TABLE 27 hPBMC hPBMC hPBMC TNFα IC₅₀ IL-1β IC₅₀ IL-6 IC₅₀ Compound (μM)(μM) (μM) (P)-ATI-450 0.004 0.012 0.145 (M)-ATI-450 >10,000 >10,000>10,000

Although embodiments herein have been described in considerable detailwith reference to certain preferred embodiments thereof, other versionsare possible. Therefore, the spirit and scope of the appended claimsshould not be limited to the description and the preferred versionscontained within this specification.

1. A method for treating an inflammatory condition, said methodcomprising: administering, to a human subject having an inflammatorycondition, an oral dose of 5 mg/day to 300 mg/day of Compound I

or a derivative thereof to treat said inflammatory condition.
 2. Themethod of claim 1, wherein 100 mg/day of the compound is administered tosaid subject.
 3. The method of claim 1, wherein 160 mg/day of thecompound is administered to said subject.
 4. The method of claim 1,wherein 200 mg/day of the compound is administered to said subject. 5.The method of claim 1, wherein 240 mg/day of the compound isadministered to said subject.
 6. The method of any claim 1, whereinCompound I comprises the Compound (P)-I

and the Compound (M)-I

in a molar ratio of (P)-I to (M)-I of about 4:1.
 7. The method of claim1, wherein Compound I comprises Compound (P)-I substantially free ofCompound (M)-I.
 8. The method of claim 6, wherein Compound (P)-I is afree base.
 9. The method of claim 8, wherein Compound (P)-I is acrystalline form.
 10. The method of claim 9, wherein the crystallineform of Compound (P)-I is crystalline Form A characterized by an PXRDpattern having a peak expressed in degrees 2θ at about 9.78±0.2.
 11. Themethod of claim 1, wherein the inflammatory condition is selected fromrheumatoid arthritis, psoriatic arthritis, cryopyrin-associate periodicsyndrome, and Hidradenitis suppurativa.
 12. The method of claim 1,wherein the inflammatory condition is selected from the group consistingof psoriasis, juvenile idiopathic arthritis, ulcerative colitis, Crohn'sdisease, ankylosing spondylitis, pancreatic cancer, metastatic breastcancer, gout, recurrent pericarditis, and idiopathic pulmonary fibrosis.13. The method of claim 1, wherein the inflammatory condition isrheumatoid arthritis and said administering is carried out underconditions effective to inhibit progression of joint damage, improvesynovitis, or both in said subject as assessed by magnetic resonanceimaging (MRD.
 14. The method of claim 1, wherein said administering iscarried out under conditions effective to significantly reduce in vivoserum levels of one or more inflammatory cytokines as compared to invivo serum levels of the one or more inflammatory cytokines in a subjectadministered a placebo.
 15. The method of claim 14, wherein the one ormore inflammatory cytokines is selected from the group consisting ofTNF-α, IL-1β, IL-6, IL-8, IFNγ, IL-17, IL-18, IL-1α, and IL-1RA.
 16. Themethod of claim 1, wherein administering further comprises:administering one or more additional therapeutic agents in conjunctionwith Compound I.
 17. The method of claim 16, wherein the one or moreadditional therapeutic agents is selected from the group consisting ofan anti-inflammatory drug, an anti-atherosclerotic drug, animmunosuppressive drug, an immunomodulatory drug, a cytostatic drug, anangiogenesis inhibitor, a kinase inhibitor, a cytokine blocker, and aninhibitor of cell adhesion molecules.
 18. The method of claim 1, whereinCompound I is formulated as a solid dosage form selected from a tablet,a capsule, a lozenge, a sachet, a powder, granules, and orallydispersible film.
 19. The method of claim 18, wherein the solid dosageform is a tablet.